Therapeutic agents comprising pro-apoptotic proteins

ABSTRACT

The present invention relates to targeted killing of a cell utilizing a chimeric polypeptide comprising a cell-specific targeting moiety and a signal transduction pathway factor. In a preferred embodiment, the signal transduction pathway factor is an apoptosis-inducing factor, such as granzyme B, granzyme A, or Bax.

The present invention claims priority to U.S. Provisional PatentApplication Ser. No. 60/306,091, filed Jul. 17, 2001; to U.S.Provisional Patent Application Ser. No. 60/332,886, filed Nov. 6, 2001;and to U.S. Provisional Patent Application Ser. No. 60/360,361, filedFeb. 28, 2002, all of which are incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The present invention is directed to the fields of cellular andmolecular biology and cancer biology. More particularly, the presentinvention provides methods and compositions concerning therapeuticagents comprising a pro-apoptosis moiety and a cell-specific targetingmoiety.

BACKGROUND OF THE INVENTION

The selective destruction of an individual cell is often desirable in avariety of clinical settings. A multitude of signal transductionpathways in the cell are linked to its death and survival, and deliveryof a limiting and/or crucial component of the pathway can be productivein terms of its destruction. A classic example of such a signaltransduction pathway is apoptosis, and a variety of elements ofapoptotic pathways would be useful to target a cell for death.Apoptosis, or programmed cell death, is a fundamental processcontrolling normal tissue homeostasis by regulating a balance betweencell proliferation and death (Vaux et al., 1994; Jacobson et al., 1997).

The serine protease granzyme B (GrB) (Lobe et al., 1986; Schmid andWeissman, 1987; Trapani et al., 1988) is integrally involved inapoptotic cell death induced in target cells upon their exposure to thecontents of lysosome-like cytoplasmic granules (or cytolytic granules)found in cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells(Henkart, 1985; Young and Cohn, 1986; Smyth and Trapani, 1995).Cytotoxic lymphocyte granules contain perforin, a pore-forming protein,and a family of serine proteases, termed granzymes (Table 1). Perforinhas some structural and functional resemblance to the complementproteins C6, C7, C8 and C9, members of complement membrane attackcomplex (Shinkai et al., 1988). In lymphocyte-mediated cytolysis,perforin is inserted into the target cell membranes and appears topolymerize to form pores (Podack, 1992; Yagita et al., 1992), whichmediates access of granzyme B to the target cell cytoplasm. Once inside,granzyme B induces apoptosis by directly activating caspases andinducing rapid DNA fragmentation (Shi et al., 1992). TABLE 1 GRANZYMES(LYMPHOCYTE SERINE PROTEASES) Enzyme Names Species Other Names ActivityA Mouse Hanukah factor, MTSP, SE-1, CTLA-3 Tryptase Rat RNKP-2,fragmentin 1 Human Hanukah factor, HTSP-1, granzyme 1 B Mouse CCP-1,CTLA-1 Asp-ase Rat Fragmentin 2, RNKP-1 Human HLP, granzyme 2, HSE26.1,CSPB C Mouse CCP-2 Unknown Rat RNKP-4 D Mouse CCP-5 Unknown E MouseCCP-3, MCSP2 Unknown F Mouse CCP-4, MCSP3 Unknown G Mouse MCSP1 UnknownH Human CCP-X, CSP-C Chymase I Rat GLP I and II Unknown J Rat RNKP-5Unknown K Rat Tryptase 2, fragmentin 3 Tryptase Human Granzyme 3Tryptase M Rat RNK-Met-1 Met-ase Human Met-aseThe granzymes are structurally related, but have diverse substratepreference. Through its unique ability to cleave after aspartateresidues, granzyme B can cleave many procaspases in vitro, and it hasbeen an important tool in analyzing the maturation of caspase-3 (Darmonet al., 1995; Quan et al., 1996; Martin et al., 1996), caspase-7(Chinnaiyan et al., 1996; Gu et al., 1996; Fernandes-Alnemri et al.,1995), caspase-6 (Orth et al., 1996; Femandes-Alnemri et al., 1995),caspase-8 (Muzio et al., 1996), caspase-9 (Duan et al., 1996), andcaspase-10a/b (Fernandes-Alnemri et al., 1996; Vincenz and Dixit, 1997).Furthermore, it is highly toxic to target cells (Shi et al., 1992). Ithas been assumed until now that granzyme B kills cells by direct caspaseactivated, supplemented under certain circumstances by direct damage todownstream caspase substrates (Andrade et al., 1998). Having gainedaccess to the cytosol, granzyme B is rapidly translocated to the nucleus(Jans et al., 1996; Trapani et al., 1996) and can cleave poly(ADP-ribose) polymerase and nuclear matrix antigen, sometimes usingdifferent cleavage sites than those preferred by caspases (Andrade etal., 1998). Although many procaspases are efficiently cleaved in vitro,granzyme B-induced caspase activation occurs in a hierarchical manner inintact cells, commencing at the level of executioner caspases such ascaspase-3, followed by caspase-7 (Yang et al., 1998). This is incontrast to FasL-mediated killing, which relies on a membrane signalgenerated through apical caspases such as caspase-8 (Muzio et al., 1996;Sarin et al., 1997). In addition, some studies showed that granzyme Bcan also induce death through a caspase-independent mechanism thatinvolves direct damage to nonnuclear structures, although the keysubstrates in this pathway have yet to be elucidated (Sarin et al.,1997; Trapani et al., 1998; Heibein et al., 1999; Beresford et al.,1999).

Studies by Froelich and co-workers suggest that GrB is internalized byreceptor-mediated endocytosis, and that the role of perforin is tomediate release of granzyme B from endocytic vesicles. In fact, perforincan be replaced by other vesicle-disrupting factors such as thoseproduced by adenovirus (Froelich et al., 1996; Pinkoski et al., 1998;Browne et al., 1999).

Granzymes in general are highly homologous, with 38-67% homology to GrB(Haddad et al., 1991), and they contain the catalytic triad (His-57,Asp-102, and Ser-195) of trypsin family serine proteases. Other featuresinclude the mature, N-terminal Ile-Ile-Gly-Gly sequence, three or fourdisulfide bridges, and a conserved motif (PHSRPYMA), which also appearsin neutrophil cathepsin G and mast cell chymases. The carbohydratemoieties of granzymes are Asn-linked (Griffiths and Isaaz, 1993). Thegranzyme mRNA transcripts are translated as pre-pro-proteases. The pre-or leader sequence is cleaved by signal peptidase at the endoplasmicreticulum. When the propeptides are removed, the inactive progranzymes(zymogens) become active proteases. The granzyme propeptides sequencesstart after the leader peptide and end before the N-terminal Ile neededfor the protease to fold into a catalytic conformation (Kam et al.,2000).

Among the various apoptotic factors identified so far, members of theBcl-2 family represent some of the most well-defined regulators of thisdeath pathway. Some members of the Bcl-2 family, including Bcl-2,Bcl-XL, Ced-9, Bcl-w and so forth, promote cell survival, while othermembers including Bax, Bcl-Xs, Bad, Bak, Bid, Bik and Bim have beenshown to potentiate apoptosis (Adams and Cory, 1998). A number ofdiverse hypotheses have been proposed so far regarding the possiblebiological functions of the Bcl-2 family members. These include dimerformation (Oltvai et al., 1993), protease activation (Chinnaiyan et al.,1996), mitochondrial membrane depolarization (6), generation of reactiveoxygen intermediates (Hockenbery et al., 1993), regulation of calciumflux (Lam et al., 1994; Huiling et al., 1997), and pore formation(Antonsson et al., 1997; Marzo et al., 1998).

Bax, a 21 kDa death-promoting member of the Bcl-2 family, was firstidentified as a protein that co-immunoprecipated with Bcl-2 fromdifferent cell lines (Oltvai et al., 1993). Overexpression of Baxaccelerates cell death in response to a wide range of cytotoxic results.Determination of the amino acid sequence of the Bax protein showed it tobe highly homologous to Bcl-2. The Bax gene consists of six exons andproduces alternative transcripts, the predominant form of which encodesa 1.0 kb mRNA and is designated Baxα. Like Bcl-2 and several othermembers of the Bcl-2 family, the Bax protein has highly conservedregions, BH1, BH2 and BH3 domains, and hydropathy analysis of thesequences of these proteins indicates the presence of a hydrophobictransmembrane segment at their C-terminal ends (Oltvai et al., 1993).

Bax is widely expressed without any apparent tissue specificity.However, on the induction of apoptosis, Bax translocates intomitochondria, resulting in mitochondria dysfunction and release ofcytochrome c, which subsequently activates caspase pathways (Hsu andYoule, 1997; Wolter et al., 1997; Gross et al., 1998). Thistranslocation process is rapid and occurs at an early stage of apoptosis(Wolter et al., 1997). Selective overexpression of Bax in human ovariancancer through adenoviral gene transfer resulted in significant tumorcell kill in vivo (Tai et al., 1999). Overexpression of the Bax gene bya binary adenovirus system in cultured cell lines from human lungcarcinoma results in caspase activation, apoptosis induction, and cellgrowth suppression. Moreover, intratumoral injection of adenovirusvector expressing the Bax gene suppressed growth of human lung cancerxenografts established in nude mice (Kagawa et al., 2000; Kagawa et al.,2000).

WO 99/45128 and Aqeilan et al. (1999) are directed to chimeric proteinshaving cell-targeting specificity and apoptosis-inducing activities,particularly the recombinant chimeric protein IL-2-Bax, whichspecifically targets IL2 receptor-expressing cells and inducescell-specific apoptosis.

WO 99/49059 relates to a chimeric toxin comprised of gonadotropinreleasing hormone (GnRH) and Pseudomonas exotoxin A (PE) to detect atumor-associated epitope expressed by human adenocarcinoma.

WO 97/46259 concerns targeted chimeric toxins comprising cell targetingmoieties and cell killing moieties directed to neoplastic cells. In aspecific example, the chimeric toxin comprises gonadotropin releasinghormone homologs and Pseudomonas Exotoxin A.

WO 97/22364 addresses targeted treatment of allergy responses, whereby achimeric cytotoxin Fc_(2′-3)-PE₄₀ is directed to targeted elimination ofcells expressing the FcεRI receptor.

While some chimeric protein compositions have been described, othermethods and compositions are needed for improved therapies involving thekilling of cells.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions involvingthe delivery of chimeric polypeptides comprising signal transductionpathway factors that induce death of a targeted cell. In a preferredembodiment, this factor is a pro-apoptotic factor.

Almost all cells contain mechanisms responsible for mediating cell-death(apoptosis). Thus, in some embodiments, the present invention addressesdelivery of certain pro-apoptotic proteins that are central mediators ofthis effect to the interior of target cells, which will result in celldeath through apoptotic mechanisms. The apoptosis-inducing moietyinduces programmed cell death upon entry into the target cell of thechimeric polypeptide, which is delivered for binding to the target cellby the cell-specific targeting moiety. In some embodiments of thepresent invention and as an advantage over known methods in the art,pro-apoptotic polypeptides are delivered as proteins and not as nucleicacid molecules to be translated to produce the desired polypeptides. Asan additional advantage, human sequences are utilized in the chimericpolypeptides of the present invention to circumvent any undesirableimmune responses from a foreign polypeptide.

In further embodiments, granzyme A or granzyme B is a mediator forinducing apoptosis. In specific embodiments, recombinant ligand (VEGF)and/or recombinant antibody (scFvMEL) moieties are fused as nucleic acidsequences to those sequences that encode a granzyme or a Bcl-2 familymember. The inventors present data herein demonstrating that chimericpolypeptides, such as granzymeB-vegf121 and granzymeB-scFvMEL, arecytotoxic to target cells. Given that a skilled artisan recognizes thatthere are multiple similar cell-targeting and pro-apoptotic examplesthat may be used interchangeably with the specific examples herein, thisindicates that constructs containing pro-apoptotic proteins havesignificant therapeutic potential for the treatment of disease statesand represent a new class of therapeutic agents with a novel mechanismof action.

In an embodiment in which pro-apoptotic proteins are utilized as thekilling moiety in chimeric proteins, recombinant antibody (scFvMEL) thatbinds to the cell-surface antigen gp240 of melanoma cells and isinternalized efficiently is utilized. The inventors fused the genesencoding scFvMEL to genes encoding Bax, truncated Bax1-5, and Bax 345,respectively (designated as scFvMEL-bax, scFvMEL-Bax 1-5 andscFvMEL-Bax345, respectively). These genes were inserted intoprotein-expression vectors and transformed into bacteria. The fusionproteins were purified, tested against target cells in culture, andshown to be cytotoxic to target cells. This suggests that constructscontaining the pro-apoptotic protein Bax have significant therapeuticpotential for the treatment of diseases and present a new class oftherapeutic agents with a novel mechanism of action.

In an object of the present invention, there is a chimeric polypeptidecomprising a cell-specific targeting moiety and a signal transductionpathway factor.

In another object of the present invention, there is a chimericpolypeptide comprising a cell-specific targeting moiety and anapoptosis-inducing factor, wherein said apoptosis-inducing factor is agranzyme. In a specific embodiment, the granzyme is granzyme B. Inanother specific embodiment, the amino acid sequence of said granzyme Bis selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In anotherspecific embodiment, the amino acid sequence of said granzyme B is SEQID NO:60, SEQ ID NO:60 further comprising an N-terminal extension of SEQID NO:61, or SEQ ID NO:60 wherein the first twenty amino acids areabsent. In a further specific embodiment, the amino acid sequence ofsaid granzyme B is at least 100 contiguous amino acids from SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, or SEQ ID NO:60. In a further specific embodiment, the amino acidsequence of said granzyme B is at least 75 contiguous amino acids fromSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, or SEQ ID NO:60. In a further specific embodiment, theamino acid sequence of said granzyme B is at least 40 contiguous aminoacids from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO:16, or SEQ ID NO:60. In an additional specificembodiment, the granzyme is granzyme A. In a further specificembodiment, the amino acid sequence of said granzyme A is selected fromthe group consisting of SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25. Ina further specific embodiment, the amino acid sequence of said granzymeA is at least 100 contiguous amino acids from SEQ ID NO:23, SEQ ID NO:24or SEQ ID NO:25. In a further specific embodiment, the amino acidsequence of said granzyme A is at least 75 contiguous amino acids fromSEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. In a further specificembodiment, the amino acid sequence of said granzyme A is at least 40contiguous amino acids from SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.In an additional specific embodiment, the cell-specific targeting moietyis a cytokine, an antibody, a ligand, or a hormone. In a furtherspecific embodiment, the ligand is VEGF. In a further specificembodiment, the VEGF is vegf121. In a further specific embodiment, theantibody is a single chain antibody. In a further specific embodiment,the single chain antibody is scFvMEL. In an additional specificembodiment, the granzyme is granzyme B and said cell-specific targetingmoiety is vegf121. In another specific embodiment, the granzyme isgranzyme B and said cell-specific targeting moiety is scFvMEL. In afurther specific embodiment, the polypeptide further comprises a linker,such as SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. In a specificembodiment, the polypeptide is encoded by a recombinant polynucleotide.

In an additional object of the present invention, there is an expressioncassette comprising a polynucleotide encoding a chimeric polypeptidecomprising a cell-specific targeting moiety and an apoptosis-inducingfactor, wherein said apoptosis-inducing factor is a granzyme, andwherein said polynucleotide is under control of a regulatory sequenceoperable in a host cell. In specific embodiments, the granzyme isgranzyme A or granzyme B. In a specific embodiment, the granzyme A isencoded by a polynucleotide of SEQ ID NO:26, SEQ ID NO:27, or SEQ IDNO:28. In another specific embodiment, the granzyme B is encoded by apolynucleotide of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, or SEQ ID NO:22. In an additional specificembodiment, the cassette is comprised in a recombinant viral vector,such as an adenoviral vector, an adeno-associated viral vector, or aretroviral vector.

In an additional object of the present invention, there is a host cellcomprising an expression cassette comprising a polynucleotide encoding achimeric polypeptide comprising a cell-specific targeting moiety and anapoptosis-inducing factor, wherein said apoptosis-inducing factor is agranzyme. In specific embodiments, the cell is further defined as aprokaryotic host cell or an eukaryotic host cell.

In another object of the present invention, there is a method of using ahost cell comprising an expression cassette comprising a polynucleotideencoding a chimeric polypeptide comprising a cell-specific targetingmoiety and an apoptosis-inducing factor, wherein said apoptosis-inducingfactor is a granzyme, comprising culturing the host cell underconditions suitable for the expression of the chimeric polypeptide.

In an additional object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a granzyme. In specific embodiments, the granzymeis granzyme A or granzyme B. In specific embodiments, the cell is invivo and/or in a human.

In another object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a granzyme, wherein said cell-specific targetingmoiety is scFvMEL and said granzyme is granzyme B. It is contemplatedthat the cell-specific targeting moiety acts by targeting specificcells, for example, cells that express on their surface a peptide orpolypeptide that is capable of specifically binding the targetingmoiety. The compound that allows the cell to be specifically targetedmay be referred herein as the target. Thus, in some embodiments of theinvention, cells may have a target to which the cell-specific targetingmoiety recognizes.

In another object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a granzyme, wherein said cell-specific targetingmoiety is vegf121 and said granzyme is granzyme B.

In an additional object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a pro-apoptotic member of the Bcl-2 family. In aspecific embodiment, the pro-apoptotic member of the Bcl-2 family is Baxor a fragment thereof. In specific embodiments, the cell is in vivoand/or in a human. In a specific embodiment, the fragment of Bax lacksat least part of a polypeptide encoded by exon 6 in a Bax polynucleotidesequence.

In another object of the present invention, there is a method ofinducing apoptosis in a cell, comprising administering to said cell aneffective amount of a chimeric polypeptide comprising a cell-specifictargeting moiety and a pro-apoptotic member of the Bcl-2 family, whereinsaid cell-specific targeting moiety is scFvMEL and said pro-apoptoticmember of the Bcl-2 family is Bax or a fragment of Bax. In a specificembodiment, the fragment of Bax lacks at least part of exon 6 in a Baxpolynucleotide sequence.

In an additional object of the present invention, there is a method oftreating a disease in an individual, comprising the steps ofadministering to said individual a therapeutically effective amount of acomposition comprising a chimeric polypeptide comprising anapoptosis-inducing moiety and a cell-specific targeting moiety; and apharmaceutical carrier. In a specific embodiment, the pharmaceuticalcarrier comprises a lipid. In another specific embodiment, the diseaseis cancer, diabetes, arthritis, or inflammatory bowel disease,atherosclerosis, or diabetic retinopathy. In an additional specificembodiment, the disease is cancer. In a further specific embodiment, theapoptosis-inducing moiety is a granzyme. In a further specificembodiment, the granzyme is granzyme B or a fragment thereof. In anadditional specific embodiment, the apoptosis-inducing moiety is apro-apoptotic member of the Bcl-2 family. In another specificembodiment, the pro-apoptotic member of the Bcl-2 family is Bax or afragment thereof. In an additional specific embodiment, the fragment ofBax lacks at least part of a polypeptide encoded by exon 6 in a Baxpolynucleotide sequence. In another specific embodiment, the fragment ofBax lacks at least part of a polypeptide encoded by exons selected fromthe group consisting of 4, 5, and 6. In an additional specificembodiment, the administration is by intravenous injection. In anotherspecific embodiment, the administration is by inhalation. In a furtherspecific embodiment, the administration is intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally, byinhalation (e.g. aerosol inhalation), by injection, by infusion, bycontinuous infusion, by localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in a creme, or in a lipidcomposition. In a specific embodiment, the method further comprisesadministering to said individual an anti-inflammatory composition,chemotherapy, surgery, radiation, hormone therapy, or gene therapy.

It is contemplated that aspects of the invention discussed in thecontext of one embodiment of the invention may be employed with respectto any other embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 illustrates human pre-mature granzyme B cDNA from Hut78 cells. A1% agarose gel electrophoresis demonstrates human pre-mature granzyme BcDNA synthesized from Hut78 cells by RT-PCR. Lane 1 represents a lowmass DNA molecular marker; lane 2 represents control synthesized cDNA(˜500 bp); lane 3 represents no RT control; and lane 4 represents humanpre-mature granzyme B cDNA (˜800 bp).

FIG. 2 shows the nucleotide sequence encoding human pre-mature granzymeB (SEQ ID NO:54) and amino acid sequence (SEQ ID NO:55).

FIG. 3 illustrates construction of pET32GrB-vegf121 and pET32GrB-scFvMELfusion constructs. Construction of these fusion constructs was based ona PCR method. FIG. 3A shows the construction of pET32GrB-vegf121. FIG.3B shows the construction of pET32GrB-scFvMEL. The full length geneswere ligated into the Xba I/Xho I site of the expression vectorpET-32a(+).

FIG. 4 demonstrates the predicted structure of recombinant granzymeB-vegf121 (FIG. 4A), granzyme B-scFvMEL (FIG. 4B) in pET32a vectorexpressed in E. coli and the sequences of granzyme B-vegf121 (FIGS. 4Cand 4D) (SEQ ID NO:56 for nucleic acid sequence and SEQ ID NO:57 foramino acid sequence), granzyme B-scFvMEL (FIGS. 4E and 4F) (SEQ ID NO:58for nucleic acid sequence and SEQ ID NO:59 for amino acid sequence). ThepET32a(+) vector contains a T7 promoter for high-level expression.Expression of the nucleic acid includes sequence containing the Trx.tag,followed by a His.tag, a thrombin cleavage site, and an enterokinasecleavage site for final removal of the protein purification tag.

FIG. 5 shows SDS-PAGE analysis of the expression of the fusion proteinsSDS-PAGE Coomassie Blue staining of granzyme B-vegf121 (FIG. 5A) andgranzyme B-scFvMEL (FIG. 5B) under reducing conditions. Panel A of FIG.5A shows SDS-PAGE Coomassie Blue staining of Granzyme B-vegf121. Lane 1shows non-induced total cell lysates; lane 2 shows induced total celllysates; lane 3 shows non-induced soluble; lane 4 shows induced soluble;lane 5 shows non-induced insoluble; lane 6 shows induced insoluble; lane7 shows protein molecular marker. In Panel B, lane 1 shows proteinmolecular marker; lane 2 shows pro-granzyme B-vegf121 (IMAC-eluate fromTalon Resin); lane 3 shows pro-granzyme B-vegf121 (IMAC-Elute fromNickel NTA), Lane 4: Granzyme B-vegf121 (after rEK cut). In FIG. 5B,there is shown SDS-PAGE Coomassie blue staining of granzyme B-scFvMEL.In Panel C, lane 1 shows protein molecular marker; lane 2 showsnon-induced total cell lysates; lane 3 shows induced total cell lysates;lane 4 shows non-induced soluble; lane 5 shows induced soluble; lane 6shows non-induced insoluble; lane 7 shows induced insoluble. In Panel D,lane 1 shows protein molecular marker; lane 2 shows pro-granzymeB-scFvMEL (IMAC-eluate from Nickel NTA); lane 3 shows granzyme B-scFvMEL(after rEK cut).

FIG. 6 demonstrates a Western blot analysis of granzyme B-vegf121 andgranzyme B-scFvMEL.

FIG. 7 shows binding activity of scFvMEL moiety of granzyme B-scFvMELfusion protein. ELISA of different scFvMEL fusion proteins were examinedon a plate pre-coated with Protein L.

FIG. 8 demonstrates testing of cytotoxicity of granzyme B-vegf121against log-phase PAE-Flk-1 and PAE-Flt-1

FIG. 9 demonstrates testing of cytotoxicity of granzyme B-scFvMEL onA375-M.

FIG. 10 illustrates the human Bax gene, its exons, and domains BH1, BH2,and BH3.

FIG. 11 demonstrates cloning of human Bax cDNA from Namalwa cells byPCR. Lane 1: Low Mass DNA Molecular Marker, lanes 2-6: Controlsynthesized cDNA (˜500 bp), lanes 7-8: Human Bax cDNA (˜580 bp) usingrandom primer (lane 7) and using Oligo(dT) primer (lane 8).

FIGS. 12A and 12B illustrate construction of scFvMEL-bax-related fusionconstructs.

FIG. 13 shows SDS-PAGE and Coomassie Blue Staining analysis of theexpression of the fusion proteins.

FIG. 14 shows the expression of pET32-scFvMEL-bax and pET32-Bax-scFvMELtransformed into AD494(DE3)pLysS E coli and under IPTG induction.

FIG. 15 demonstrates western blotting analysis of the expression of thefull length bax and Bax-scFvMEL proteins. Lane 1: pBad/HisA (negativecontrol), Lane 2: pBad/HisLacZ (expression positive control), Lane 3-5:Bax protein (lane 3: expression in RM+glucose+ampicillin, lane 4:expression in RM+ampicillin, lane 5: expression in LB+ampicillin), Lanes6-8: Bax-scFvMEL protein (lane 6: expression in RM+glucose+ampicillin,lane 7: expression in RM+ampicillin, lane 8: expression inLB+ampicillin).

FIGS. 16A and 16B demonstrate the binding activity of scFvMEL moiety offusion proteins.

FIG. 17 shows the cytotoxicity of scFvMEL-bax345 and Bax345-scFvMELfusion proteins on A375-M.

FIG. 18 shows ELISA of granzyme B-Vegf121 on various cell lines(detected with mouse anti-vegf121 antibody and mouse anti-granzyme Bantibody).

FIG. 19 demonstrates cytotoxicity of Granzyme B-VEGF121 on transfectedendothelial cells.

FIG. 20 shows cytotoxicity assay of granzyme B-Vegf121 vs. vegf121 rgelin vitro against PAE/FLK-1.

FIG. 21 illustrates caspase activity on PAE cells treated with GranzymeB-Vegf121.

FIG. 22 demonstrates cytochrome c release of PAE cells treated withGRB/VEGF121.

FIG. 23 shows Bax translocation of PAE cells after GRB/VEGF₁₂₁treatment.

FIG. 24 illustrates cytochrome c release in A375-M vs. SKBR3-HP cellstreated with GRB/scFvMEL.

FIG. 25 illustrates GrB/VEGF121 induces DNA laddering on PAE/flk-1cells.

FIG. 26 shows ELISA of GrB/scFvMEL on gp240 Ag-positive A375-M vs gp240Ag-negative T-24 cells detected by Grb mouse mAb.

DETAILED DESCRIPTION OF THE INVENTION

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

The term “apoptosis” as used herein is defined as programmed cell death;an endogenous cell death program results in the death of the cell.

The term “cytokine” as used herein is defined as an agent made by a cellthat affects the behavior of another cell. In a specific embodiment, theagent is a polypeptide. For example, cytokines made by lymphocytes areoften called lymphokines or interleukins (IL). Furthermore, cytokinesact on specific cytokine receptors on the cells that they affect. In aspecific embodiment, the term “cytokine” includes growth factors.

The term “granzyme” as used herein is defined as an enzyme from thegranules of cytotoxic lymphocytes that, upon entry into the cytosol of acell, induce apoptosis and/or nuclear DNA fragmentation. In a specificembodiment, the granzyme is a lymphocyte serine protease. In someembodiments, the granzyme is full-length, whereas in other embodimentsthe granzyme is partial.

The term “signal transduction pathway factor” as used herein is definedas an enzyme, substrate, cofactor or other protein which influencesbiological activity of another enzyme, cofactor or protein. In aspecific embodiment, the factor is associated with receptor-mediatedsignaling that transmits a signal from outside the cell membrane tomodulate a growth response within the cell. In one embodiment, thegrowth response that is modulated is a pro-growth response. In analternative embodiment, the growth response that is modulated is ananti-growth response, such as induction of apoptosis.

The present invention relates to chimeric proteins with cell-targetingspecificity and cell-destruction moieties, such as from signaltransduction pathways linked, either directly or indirectly, to celldeath. In some embodiments, the cell-destruction moieties areapoptosis-inducing activities. The chimeric proteins of the inventionare composed of a cell-specific targeting moiety and anapoptosis-inducing moiety. The cell-specific targeting moiety providescell-specific binding properties to the chimeric protein, while theapoptosis-inducing moiety induces programmed cell death upon entry intoa target cell. In some embodiments, the chimeric proteins of theinvention are delivered as polypeptides and are produced by recombinantexpression of a fusion polynucleotide between a coding sequence of acell-targeting moiety and a coding sequence of an apoptosis-inducingprotein. Such chimeric proteins are likely to be superior to theimmunotoxins currently used in the art because they are of human originand thus are expected to have reduced immunogenicity in a humanrecipient. In addition, chimeric proteins kill target cells by inducingapoptosis which does not cause a release of cellular organelles into theextracellular environment to result in an inflammatory response. Whencells die by the apoptotic pathway, they shrink and condense, but theorganelles and plasma membranes retain their integrity, and the deadcells are rapidly phagocytosed by neighboring cells or macrophagesbefore there is leakage of the cells' contents, thereby elicitingminimal tissue or systemic response.

The invention also relates to pharmaceutical compositions of thechimeric proteins, methods of producing such proteins and methods ofusing the same in vitro and in vivo, especially for eliminating specificundesirable target cells, and for the treatment of a variety of diseaseconditions as well as the use of the proteins for disease diagnosis.

In the present invention, methods and compositions regarding targeteddestruction of a cell utilizing a chimeric polypeptide are disclosed.The chimeric polypeptide is comprised of at least two moieties: onemoiety is the effectual component for killing of the cell; the secondmoiety is the delivery component of the chimeric polypeptide to targetthe killing component to the cell of interest. In some embodiments ofthe present invention, at least one of the moieties, and preferablyboth, are of human origin, which eliminates an immune response from theindividual to whom the chimeric polypeptide is administered. In oneembodiment, the moiety for killing the cell is a component of a signaltransduction pathway, such as one which is a limiting factor orrestriction point in the pathway. Delivery of the signal transductionpathway bypasses the requirement to elicit upstream steps of thepathway, and the resultant administration of this restriction point endsin the same effect, which is destruction of the cell. A skilled artisanrecognizes that types of agents which could be delivered intracellularlyto mediate signal transduction include enzymes, such as kinases (forexample, protein kinase B (PKB, AKT), which mediates insulin signaling;protein kinase C, which is involved in numerous signaling events; andphosphatidylinositol 3-kinase, which is involved in numerous signalingevents); phosphatases; proteases (such as caspase 3); nucleases (such ascaspase-activated deoxyribonuclease (CAD), which is a mediator ofapoptosis); phospholipases; NCKAP1 (which is an apoptosis-relatedprotein down-regulated in the brain tissues of Alzheimer's patients;Suzuki et al., 2000) or co-factors, such as cytochrome c (which isinvolved in apoptosis signaling) and cyclic AMP (which is involved innumerous pathways).

In a specific embodiment, the signal transduction pathway factor is anenzyme. The enzyme may be a hydrolase (e.g., deaminase, esterase,glycosidase, lipase, nuclease, peptidase, phosphatase,phosphodiesterase, and proteinase); isomerase (e.g., epimerase, mutase,and racemase); ligase or synthetase (e.g., acyl-CoA synthetase,amino-acyl-tRNA synthetase, and carboxylase); lyase (e.g., aldolase,decarboxylase, dehydratase, and nucleotide cyclase); oxidoreductase(e.g., dehydrogenase, dioxygenase, hydrogenase, monooxygenase,nitrogenase, oxidase, and reductase); and/or transferase (e.g.,acyltransferase, aminotransferase, glycosyltransferase, kinase,methyltransferase, nucleotidyltransferase, phosphorylase, andsulphotransferase). In specific embodiments, the enzyme is classified asa toxin, which means it is toxic to a cell, tissue, or organism.

In some embodiments, the signal transduction pathway factor is anapoptosis-inducing factor. Almost all cells contain mechanismsresponsible for mediating cell death (apoptosis). In a specificembodiment, and as demonstrated in the Examples herein, delivery ofgranzyme B protein into the interior of target cells results in celldeath through apoptotic mechanisms. Using recombinant ligand (VEGF) andrecombinant antibody (scFvMEL), which bind to the cell-surface of tumorcells and internalize efficiently, the inventors designed two novelgranzyme B-related fusion proteins: GrB-vegf121 to specifically targetthe endothelial cells; and GrB-scFvMEL to specifically target themelanoma cells.

A skilled artisan recognizes particular cell-specific targeting moietieswhich would be useful in the chimeric polypeptide to target a cell ofinterest. For example, the cell-specific targeting moieties may beantibodies to a particular cell marker(s), growth factor(s), hormone(s),or cytokine(s).

A skilled artisan is aware that nucleic acid sequences and amino acidsequences useful for generating the chimeric polypeptide of the presentinvention are readily obtainable, particularly through public databases,such as the National Center for Biotechnology Information's (NCBI)GenBank database, or commercially available databases such as fromCelera Genomics, Inc. (Rockville, Md.). For example, granzyme B aminoacid sequences useful in the present invention may include, followed bytheir GenBank Accession number, at least: P10144 (SEQ ID NO:11);XP_(—)012328 (SEQ ID NO:12); A61021 (SEQ ID NO:13); NP_(—)004122 (SEQ IDNO:14); CAA01810 (SEQ ID NO:15); and/or AAA75490 (SEQ ID NO:16). SEQ IDNO:60 is human granzyme B sequence reflecting variances seen in SEQ IDNO:11 through SEQ ID NO:16, such as at residue 55 (a Gln or an Arg), atresidue 94 (a Pro or an Ala), as an N-terminal extension comprising SEQID NO:61 (MKSLSLLHLFPLPRAKREQGGNNSSSNQGSLPEK), and/or as a deletion ofresidues 1 through 20.

Granzyme B nucleic acid sequences useful in the present invention mayinclude at least: XM_(—)012328 (SEQ ID NO:17); BF589964 (SEQ ID NO:18);BF221604 (SEQ ID NO:19); NM_(—)004131 (SEQ ID NO:20); A26437 (SEQ IDNO:21); and/or M28879 (SEQ ID NO:22). Granzyme A amino acid sequencesuseful in the present invention may include, followed by their GenBankAccession number, at least: P12544 or NP_(—)006135 (SEQ ID NO:23) orXP_(—)003652 (SEQ ID NO:24). SEQ ID NO:25 comprises a human granzyme Aamino acid sequence and reflects variance in SEQ ID NO:23 and SEQ IDNO:24 at residue 121 (Thr or Met, respectively). Granzyme A nucleic acidsequences useful in the present invention may include at least:XM_(—)003652 (SEQ ID NO:26); NM_(—)006144 (SEQ ID NO:27); and/or U40006(SEQ ID NO:28). A skilled artisan recognizes how to retrieve these andrelated sequences from the NCBI GenBank database.

I. Apoptosis-Inducing Proteins

Strictly regulated cell death is required for the development ofmultilineage organisms and the maintenance of homeostasis withintissues. Differentiation status of an individual cell directly affectswhether it can execute a suicidal response following a death stimulusvaries. Both positive and negative regulators of programmed cell death(apoptosis) have been identified. Bcl-2 is a repressor of programmedcell death (Vaux et al., 1988), and recently, other Bcl-2 homologueswere shown to inhibit apoptosis. However, one homolog of Bcl-2, Bax,mediates an opposite effect through acceleration of apoptosis. In theBcl-2 family there is notable homology clustered within two conservedregions: Bcl-2 homology domains 1 and 2 (BH1 and BH2) (Oltvai et al.,1993; Boise et al., 1993; Kozopas et al., 1993; Lin et al., 1993).Members of the Bc1 family include Bax, Bc1-X_(L), Mc1-1, A1 and severalopen reading frames in DNA viruses. Another conserved domain in Bax,distinct from BH1 and BH2, is termed BH3 and mediates cell death andprotein binding functions (Chittenden et al., 1995). A subset of thepro-apoptotic proteins contains only the BH3 domain, implying that thisparticular domain may be uniquely important in the promotion ofapoptosis (Diaz et al., 1997).

In vivo Bax homodimerizes and also forms heterodimers with BCL-2, andoverexpressed Bax overrides the death repressor activity of BCL-2(Oltvai et al., 1993). Bax expression levels higher than Bcl-2expression levels in bladder tumors correlates to an improved patientprognosis. In patients whose tumors expressed more Bcl-2 than Bax mRNA,early relapses were much more frequently observed (Gazzaniga et al.,1996).

Recently it was reported that a splice variant of Bax, Bax-alpha, wasexpressed in high amount in normal breast epithelium, whereas only weakor no expression was detected in 39 out of 40 cancer tissue samplesexamined (Bargou et al., 1996), and downregulation of Bax-alpha wasdetected in different histological subtypes. Furthermore, when Bax-alphawas transfected into breast cancer cell lines under the control of atetracycline-dependent expression system, Bax restored sensitivity ofthe cancer cells toward both serum starvation and APO-I/Fas-triggeredapoptosis, significantly reducing tumor growth in SCID mice. Therefore,it was proposed that disruption of apoptosis pathway may contribute tothe pathogenesis of breast cancer at least in part due to an imbalancebetween members of the Bcl-2 gene family (Bargou et al., 1996).

Additional members of the Bcl-2 family of apoptosis-inducing proteinshave been identified. Bak, a new member of the Bcl-2 family, isexpressed in a wide variety of cell types and binds to the Bcl-2homologue Bcl-x2 in yeast (Farrow et al., 1995; Chittenden et al.,1995). A domain in Bak was identified as both necessary and sufficientfor cytotoxicity activity and binding to Bcl-x1. Furthermore, sequencessimilar to this domain that are distinct from BH1 and BH2 have beenidentified in Bax and Bip1. This domain is critical for mediating thefunction of multiple cell death-regulatory proteins that interact withBcl-2 family members (Chittenden et al., 1995).

Overexpression of Bak in sympathetic neurons deprived of nerve growthfactor accelerated apoptosis and blocked the protective effect ofco-injected E1B 19K. The adenovirus E1B 19K protein is known to inhibitapoptosis induced by E1A, tumor-necrosis factor-alpha, FAS antigen andnerve growth factor deprivation (Farrow et al., 1995). Expression of Bakinduced rapid and extensive apoptosis of serum-deprived fibroblasts,which suggests that Bak is directly involved in activating the celldeath machinery (Chittenden et al., 1995). In the normal and neoplasticcolon, mucosal expression of immunoreactive Bak co-localized with sitesof epithelial cell apoptosis. Induction of apoptosis in the human coloncancer cell line HT29 and the rat normal small intestinal cell line 1EC18 in culture was accompanied by increased Bak expression withoutconsistent changes in expression of other Bcl-2 homologous proteins(Moss et al., 1996). Therefore, Bak was also suggested to be theendogenous Bcl-2 family member best correlated with intestinal cellapoptosis (Moss et al., 1996).

Unlike Bax, however, Bak can inhibit cell death in anEpstein-Barr-virus-transformed cell line. Tissues with uniquedistribution of Bak messenger RNA include those containing long-lived,terminally differentiated cell types (Krajewski, et al., 1996),suggesting that cell-death-inducing activity is broadly distributed, andthat tissue-specific modulation of apoptosis is controlled primarily byregulation of molecules that inhibit apoptosis (Kiefer et al., 1995).

Another member of the Bcl2 family, Bad, possesses the key amino acidmotifs of BH1 and BH2 domains. Bad lacks the classical C-terminalsignal-anchor sequence responsible for the integral membrane positionsof other family members. Bad selectively dimerizes with Bcl-x_(L) aswell as Bcl-2, but not with Bax, Bcl-Xs-Mcl1, A1 or itself. Bad reversesthe death repressor activity of Bcl-X_(L), but not that of Bcl-2 (Yanget al., 1995; Ottilie et al., 1997; Zha et al., 1997).

Bik, another member of the Bcl-2 family, interacts with the cellularsurvival-promoting proteins, Bcl-2 and Bcl-X_(L) as well as the viralsurvival-promoting proteins, Epstein Barr virus-BHRF1 and adenovirusE1B-19 kDa. In transient transfection assays, Bik promotes cell death ina manner similar to Bax and Bak, other pro-apoptotic members of theBcl-2 family. This pro-apoptosis activity of Bik can be suppressed bycoexpression of Bcl-2, Bcl-X_(L), EBV-BHRF1 and E1B-19 kDa proteins,which suggests that Bik may be a common target for both cellular andviral anti-apoptotic proteins. While Bik does not contain overt homologyto the BH1 and BH2 conserved domains characteristic of the Bcl-2 family,it shares a 9 amino acid domain (BH3) with Bax and Bak, which may be acritical determinant for the death-promoting activity of these proteins(Boyd et al., 1995; Han et al., 1996).

The Bcl-2 family is composed of various pairs of antagonist and agonistproteins that regulate apoptosis, although whether their function isinterdependent remains unclear. Utilizing gain-and loss of-functionmodels of Bcl-2 and Bax, Knudson et al. (1997), demonstrated thatapoptosis and thymic hypoplasia, characteristic of Bcl-2-deficient mice,are largely absent in mice also deficient in Bax. A single copy of Baxpromoted apoptosis in the absence of Bcl-2. However, overexpression ofBcl-2 still repressed apoptosis in the absence of Bax. While an in vivocompetition exists between Bax and Bcl-2, each is able to regulateapoptosis independently. Bax has been shown to form channels in lipidmembranes and trigger the release of liposome-encapsulatedcarboxyluorescein at both neutral and acidic pH. At physiological pH,release could be blocked by Bcl-2. In planer lipid bilayers, Bax formedpH- and voltage-dependent ion-conduction channels. Thus, thepro-apoptotic effects of Bax may be elicited through an intrinsicpore-forming activity that can be antagonized by Bcl-2 (Antonsson etal., 1997). Two other members of this family, Bcl-2 and Bcl-1, were alsoshown to form pores in lipid membranes (Schendel et al., 1997).

II. Granzyme B and Apoptosis

Host defenses against viruses, parasitic agents, and transformed cellsrequire cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells(Berke, 1995; Kagi et al., 1996), which induce apoptosis in target cellsusing at least two separate mechanisms. In the first mechanism, there isstimulation of cell surface death receptors (such as Fas) on the targetcells by death ligands expressed on the surface of the effector cell(Nagata and Golstein, 1995; Ashkenazi and Dixit, 1998), which then leadsto activation of caspase cascades in the target cell. In the secondmechanism, denoted “granule exocytosis,” there is vectoral transfer ofthe contents of effector cell cytoplasmic granules into the target cell(Doherty, 1993; Shresta et al., 1995a; Shresta et al., 1995b). Perforinand the granzyme family of serine proteases are important components ofthese granules.

Perform is a 70 kDa protein that binds in a calcium-dependent manner tomembrane phosphorylcholine groups (Masson and Tshopp, 1985; Young etal., 1986; Tschopp et al., 1989). Subsequent to binding, perforininserts into the membrane and oligomerizes, resulting in the formationof pores. This permeabilization of the membrane likely makes possiblethe entry of other molecules, such as granzymes, into the target cell.

Granzymes A and B are particularly abundant (Smyth et al., 1996) withinthe granules of CTLs and NK cells. Granzyme B, which is also calledfragmentin or cytotoxic T cell protease (CCP), is similar to caspaseshaving the characteristic of cleaving substrate proteins after aspartateresidues (Zunino et al., 1990; Lobe et al., 1986; Odake et al., 1991;Poe et al., 1991; Shi et al., 1992). Mice that are granzyme B knockoutsdemonstrate an important role for granzyme B in the induction of targetcell apoptosis. CTLs and NK cells derived from granzyme B^(−/−) micehave a severely reduced capacity to induce apoptotic DNA fragmentationin target cells (Shresta et al., 1995a; Heusel et al., 1994). Althoughearlier complementary studies showed that purified granzyme B alone didnot promote apoptosis when added to target cells, cotreatment withpurified granzyme B and perforin proteins induced marked DNAfragmentation and apoptotic features in four lymphoma target cell lines(Shi et al., 1992). Therefore, it is possible that granzyme B gainsentry into target cells through perforin-generated pores, although thisis controversial. Several studies have shown that granzyme B isinternalized by target cells in the absence of added perforin (Froelichet al., 1996; Jans et al., 1996; Shi et al., 1997; Pinkoski et al.,1998; Pinkoski et al., 2000). The internalized granzyme B has beenreported to reside in the cytoplasm, (Jans et al., 1996; Shi et al.,1997) or in a novel vesicular compartment. (Pinkoski et al., 1998),although the triggering of apoptosis in cells that have internalizedgranzyme B requires further addition of perforin to the cells (Froelichet al., 1996; Jans et al., 1996; Shi et al., 1997; Pinkoski et al.,1998; Pinkoski et al., 2000). It is possible that perforin is requiredfor the release of granzyme B to the target cell from internal vesicles.Other studies have indicated that perforin facilitates translocation ofgranzyme B to the nucleus, and that nuclear localization is critical tothe ability of granzyme B to cause apoptosis (Jans et al., 1996; Shi etal., 1997; Pinkoski et al., 1998; Pinkoski et al., 2000; Pinkoski etal., 1996; Trapani et al., 1996).

Although the importance of granzyme B subcellular localization remainscontroversial, it is certain that granzyme B has the ability to affectthe caspase pathway of apoptosis. In vitro studies have shown thatgranzyme B is capable of cleaving procaspase3, -6, -7, -8m -9 and -10,(Darmon et al., 1996; Darmon et al., 1996; Martin et al., 1996; Quan etal., 1996; Fernandes-Alnemri et al., 1995; Orth et al., 1996;Fernandes-Alnemri et al., 1995; Chinnaiyan et al., 1996; Gu et al.,1996; Boldin et al., 1996; Muzio et al., 1996; Duan et al., 1996;Fernandes-Alnemri et al., 1996; Medema et al., 1997; Van de Craen etal., 1997; Talanian et al., 1997). In the case of procaspases-3, -7 and-9, granzyme B-mediated processing has been shown to generate activecaspase enzymes (Darmon et al., 1995; Quan et al., 1996; Gu et al.,1996; Duan et al., 1996). More importantly, studies with whole cellshave shown that caspases are activated in target cells followingcoincubation with granzyme B and perforin (Darmon et al., 1996; Talanianet al., 1997; Shi et al., 1996). It remains to be determined, however,which caspases are the preferred in vivo substrates for granzyme B. Inany event, it is reasonable to propose that granzyme B may promoteapoptosis simply by cleaving and activating endogenous caspases in thetarget cell.

Cleavage of the caspase substrate proteins PARP, lamin B, and U1-70 kDais also observed in cells undergoing granzyme B/perforin-mediatedapoptosis (Medema et al., 1997; Talanian et al., 1997; Shi et al., 1996;Andrade et al., 1998). These cleavage events are likely due to caspasesactivated by cleavage by granzyme B, since cleavage of all threeproteins is inhibited by 100 μM DEVD- or VAD-containing peptides, whichinhibit caspases, but not granzyme B (Darmon et al., 1996; Medema etal., 1997; Talanian et al,. 1997; Shi et al., 1996; Andrade et al.,1998). Two additional caspase substrate proteins, DNA-PK_(cs) and NuMA,are also cleaved in granzyme B/perforin-treated cells, but cleavage ofthese proteins is insensitive to DEVD or VAD peptide inhibitors (Andradeet al., 1998) Moreover, the sizes of the DNA-PK_(cs) and NuMAproteolytic fragments generated by granzyme B differ from thoseresulting from caspase cleavage, which suggests that during granzymeB-mediated apoptosis, important cellular substrates are cleaved in acaspase-independent manner. The significance of thesecaspase-independent cleavage events is unknown. However, given thatgranzyme B/perforin-mediated DNA fragmentation and apoptotic death issignificantly delayed by 100 μM DEVD/VAD, (Darmon et al., 1996; Talanianet al., 1997; Shi et al., 1996) this emphasizes the necessity forcaspase activation during this form of apoptosis.

III. Granzyme A and Apoptosis

The mature granzyme A enzyme is a disulphide cross-linked homodimer of50 kDa that cleaves substrate proteins following lysine or arginineresidues (Odake et al., 1991; Gershenfeld et al., 1986; Masson et al.,1986), and granzyme A is the most abundant protease found in thegranules of CTL cells. The mechanism of action of this protease differssignificantly from that of granzyme B, although granzyme A is capable ofinducing apoptosis after loading into target cells. Furthermore, it isthought that the role of granzyme A in CTL-induced apoptosis issignificantly more subtle than that of granzyme B. For example, micewhich are deficient in granzyme A expression (granzyme A^(−/−) mice)exhibit relatively normal CTL-mediated cytotoxicity (Andrade et al.,1998), although they are unable to clear the mouse pox virus Ectromelia(Mulbacher et al., 1996). In contrast, CTLs from granzyme B^(−/−) miceare capable of inducing target cell death only after prolongedcoincubation (Heusel et al., 1994), and, therefore, granzyme B iscritically important for rapid CTL killing. Recent experiments usingmice deficient in both granzyme A and granzyme B suggest that granzyme Adoes have some role in CTL-mediated killing. CTLs from granzymeA^(−/−)/granzyme B^(−/−) mice are unable to induce target cell DNAfragmentation, even after prolonged coincubation (Shresta et al., 1999),which indicates that granzyme A activity accounts for the ability ofgranzyme B^(−/−) CTLs to induce target cell apoptosis followingprolonged exposure. Therefore, granzyme A may allow CTLs to kill targetcells under conditions where granzyme B activity is inhibited (e.g.target cells that express granzyme B inhibitors).

In studies with recombinant proteins, coincubation of granzyme A andperforin with target cells leads to rapid (within 2 hours) accumulationof DNA single-strand breaks (Hayes et al., 1980; Beresford et al.,1999), which contrasts with the rapid degradation of DNA tooligonucleosomal-length fragments seen in cells treated with granzyme Band perforin. Granzyme A/perforin treatment also leads to nuclearcondensation (Beresford et al., 1999). These effects which occur inresponse to granzyme A are insensitive to caspase inhibitors, indicatingthat these actions of granzyme A are caspase-independent (Beresford etal., 1999). In a consistent manner, granzyme A/perforin treatment doesnot result in processing/activation of procaspase-3 or cleavage of thecaspase substrate proteins PARP, lamin B, or rho-GTPase (Beresford etal., 1999) However, granzyme B-induced DNA fragmentation is strictlydependent on the activation of caspases. Both granzyme A and granzyme B(in conjunction with perforin) also induce target cell cytolysis, bothcases of which are caspase-independent events. Thus, current evidenceindicates that granzyme B is the primary CTL mediator of target cell DNAfragmentation and apoptotic death, and that the apoptotic effects ofthis protease are mediated primarily through the activation of caspase.Alternatively, granzyme A may be more of a default or specializedmediator of target cell apoptosis, with the pathways initiated bygranzyme A being distinctly different from those initiated by granzymeB.

IV. Generation of Chimeric Molecules

While the chimeric proteins of the present invention may be produced bychemical synthetic methods or by chemical linkage between the twomoieties, it is preferred that they are produced by fusion of a codingsequence of a cell-specific targeting moiety and a coding sequence of anapoptosis-inducing protein under the control of a regulatory sequencewhich directs the expression of the fusion polynucleotide in anappropriate host cell. In preferred embodiments, each of the componentsof the chimeric protein comprise functional activity for theirrespective parts being a cell-specific targeting moiety and a signaltransduction pathway factor (such as an apoptosis-inducing protein).

The fusion of two full-length coding sequences can be achieved bymethods well known in the art of molecular biology. It is preferred thata fusion polynucleotide contain only the AUG translation initiationcodon at the 5′ end of the first coding sequence without the initiationcodon of the second coding sequence to avoid the production of twoseparate encoded products. In addition, a leader sequence may be placedat the 5′ end of the polynucleotide in order to target the expressedproduct to a specific site or compartment within a host cell tofacilitate secretion or subsequent purification after gene expression.The two coding sequences can be fused directly without any linker or byusing a flexible polylinker, such as one composed of the pentamerGly-Gly-Gly-Gly-Ser (SEQ ID NO:50) repeated 1 to 3 times. Such linkerhas been used in constructing single chain antibodies (scFv) by beinginserted between V_(H) and V_(L) (Bird et al., 1988; Huston et al.,1988). The linker is designed to enable the correct interaction betweentwo beta-sheets forming the variable region of the single chainantibody. Other linkers which may be used includeGlu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO:51)(Chaudhary et al., 1990) andLys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp(SEQ ID NO:52) (Bird et al., 1988).

A. Cell-Specific Targeting Moieties

The chimeric proteins of the invention are composed of a cell-specifictargeting moiety and an apoptosis-inducing moiety. The cell-specifictargeting moiety confers cell-type specific binding to the molecule, andit is chosen on the basis of the particular cell population to betargeted. A wide variety of proteins are suitable for use ascell-specific targeting moieties, including but not limited to, ligandsfor receptors such as growth factors, hormones and cytokines, andantibodies or antigen-binding fragments thereof.

Since a large number of cell surface receptors have been identified inhematopoietic cells of various lineages, ligands or antibodies specificfor these receptors may be used as cell-specific targeting moieties. IL2may be used as a cell-specific targeting moiety in a chimeric protein totarget IL2R⁺ cells. Alternatively, other molecules such as B7-1, B7-2and CD40 may be used to specifically target activated T cells (TheLeucocyte Antigen Facts Book, 1993, Barclay et al. (eds.), AcademicPress). Furthermore, B cells express CD19, CD40 and IL4 receptor and maybe targeted by moieties that bind these receptors, such as CD40 ligand,IL4, IL5, IL6 and CD28. The elimination of immune cells such as T cellsand B cells is particularly useful in the treatment of autoimmunity,hypersensitivity, transplantation rejection responses and in thetreatment of lymphoid tumors. Examples of autoimmune diseases aremultiple sclerosis, rheumatoid arthritis, insulin-dependent diabetesmellitus, systemic lupus erythemotisis, scleroderma, and uviatis. Morespecifically, since myelin basic protein is known to be the major targetof immune cell attack in multiple sclerosis, this protein may be used asa cell-specific targeting moiety for the treatment of multiple sclerosis(WO 97/19179; Becker et al., 1997).

Other cytokines that may be used to target specific cell subsets includethe interleukins (IL1 through IL15), granulocyte-colony stimulatingfactor, macrophage-colony stimulating factor, granulocyte-macrophagecolony stimulating factor, leukemia inhibitory factor, tumor necrosisfactor, transforming growth factor, epidermal growth factor,insulin-like growth factors, and/or fibroblast growth factor (Thompson(ed.), 1994, The Cytokine Handbook, Academic Press, San Diego).

A skilled artisan recognizes that there are a variety of knowncytokines, including hematopoietins (four-helix bundles) (such as Epo(erythropoietin), IL-2 (T-cell growth factor), IL-3 (multicolony CSF),IL-4 (BCGF-1, BSF-1), IL-5 (BCGF-2), IL-6 IL-4 (IFN-β₂, BSF-2, BCDF),IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T-cell growthfactor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM(OM, oncostatin M), and LIF (leukemia inhibitory factor)); interferons(such as IFN-γ, IFN-α, and IFN-β); immunoglobin superfamily (such asB7.1 (CD80), and B7.2 (B70, CD86)); TNF family (such as TNF-α(cachectin), TNF-β(lymphotoxin, LT, LT-α), LT-β, CD40 ligand (CD40L),Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), and4-1BBL)); and those unassigned to a particular family (such as TGF-β,IL-1α, IL-1β, IL-1 RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NKcell stimulatory factor), MIF, IL-16, IL-17 (mCTLA-8), and/or IL-18(IGIF, interferon-γ inducing factor)).

Additionally, certain cell surface molecules are highly expressed intumor cells, including hormone receptors such as human chorionicgonadotropin receptor and gonadotropin releasing hormone receptor(Nechushtan et al., 1997). Therefore, the corresponding hormones may beused as the cell-specific targeting moieties in cancer therapy.

Thus, in some embodiments of the invention, no antibodies are utilizedin the chimeric polypeptides. However, antibodies are extremelyversatile and useful cell-specific targeting moieties because they canbe generated against any cell surface antigen of interest. Monoclonalantibodies have been generated against cell surface receptors,tumor-associated antigens, and leukocyte lineage-specific markers suchas CD antigens. Antibody variable region genes can be readily isolatedfrom hybridoma cells by methods well known in the art.

Over the past few years, several monoclonal antibodies have beenapproved for therapeutic use and have achieved significant clinical andcommercial success. Much of the clinical utility of monoclonalantibodies results from the affinity and specificity with which theybind to their targets, as well as long circulating life due to theirrelatively large size. Monoclonal antibodies, however, are not wellsuited for use in indications where a short half-life is advantageous orwhere their large size inhibits them physically from reaching the areaof potential therapeutic activity.

Moreover, antibodies in their native form, consisting of two differentpolypeptide chains that need to be generated in approximately equalamounts and assembled correctly, are poor candidates for therapeuticpurposes. However, it is possible to create a single polypeptide whichcan retain the antigen binding properties of a monoclonal antibody.

Single chain antibodies (SCAs) are genetically engineered proteinsdesigned to expand on the therapeutic and diagnostic applicationspossible with monoclonal antibodies. SCAs have the binding specificityand affinity of monoclonal antibodies and, in their native form, areabout one-fifth to one-sixth of the size of a monoclonal antibody,typically giving them very short half-lives. Human SCAs offer manybenefits compared to most monoclonal antibodies, including more specificlocalization to target sites in the body, faster clearance from thebody, and a better opportunity to be used orally, intranasally,transdermally or by inhalation. In addition to these benefits,fully-human SCAs can be isolated directly from human SCA librarieswithout the need for costly and time consuming “humanization”procedures. SCAs are also readily produced through intracellularexpression (inside cells) allowing for their use in gene therapyapplications where SCA molecules act as specific inhibitors of cellfunction.

The variable regions from the heavy and light chains (VH and VL) areboth approximately 110 amino acids long. They can be linked by a 15amino acid linker with the sequence (SEQ ID NO:50)₃, which hassufficient flexibility to allow the two domains to assemble a functionalantigen binding pocket. In specific embodiments, addition of varioussignal sequences allows the scFv to be targeted to different organelleswithin the cell, or to be secreted. Addition of the light chain constantregion (Ck) allows dimerization via disulfide bonds, giving increasedstability and avidity. Thus, for a single chain Fv (scFv) SCA, althoughthe two domains of the Fv fragment are coded for by separate genes, ithas been proven possible to make a synthetic linker that enables them tobe made as a single protein chain scFv (Bird et al., 1988; Huston etal., 1988) by recombinant methods. Furthermore, they are frequently useddue to their ease of isolation from phage display libraries and theirability to recognize conserved antigens (for review, see Adams andSchier, 1999). For example, scFv is utilized to target suicide genes tocarcinoembryonic antigen (CEA)-expressing tumor cells by a retrovectordisplaying anti-CEA scFv (Kuroki et al., 2000).

Finally, the Fc portion of the heavy chain of an antibody may be used totarget Fc receptor-expressing cells such as the use of the Fc portion ofan IgE antibody to target mast cells and basophils. The use ofantibodies to target a polypeptide or peptide of interest byantibody-directed therapy or immunological-directed therapy is currentlyapproved and in use in the present therapeutic market.

Thus, it is preferred that a scFv be used as a cell-specific targetingmoiety in the present invention.

B. Apoptosis-Inducing Moieties

The pro-apoptotic proteins in the BCL2 family are particularly suitablefor use as the apoptosis-inducing moieties in the present invention.Such human proteins are expected to have reduced immunogenicity overmany immunotoxins composed of bacterial toxins. Although Bax is a usefulapoptosis-inducing moiety in one embodiment of the present invention,other members in this family are suitable for use in the presentinvention and include Bak (Farrow et al., 1995; Chittenden et al., 1995;Kiefer et al., 1995), Bcl-X_(s) (Boise et al., 1993; Fang et al., 1994),Bad (Yang et al., 1995), Bid (Wang et al., 1996), Bik (Boyd et al.,1995), Hrk (Inohara et al., 1997) and/or Bok (Hsu et al., 1997). Thenucleotide sequences encoding these proteins are known in the art andare readily obtainable from databases such as GenBank, and thus cDNAclones can be readily obtained for fusion with a coding sequence for acell-specific targeting moiety in an expression vector.

Specific domains of particular members of the Bcl-2 family have beenstudied regarding their apoptosis-inducing activities. For example, theGD domain of Bak is involved in the apoptosis function (U.S. Pat. No.5,656,725). In addition, Bax and Bipla share a homologous domain.Therefore, any biologically active domains of the Bcl-2 family may beused as an apoptosis-inducing moiety for the practice of the presentinvention.

Caspases also play a central role in apoptosis and may well constitutepart of the consensus core mechanism of apoptosis. Caspases areimplicated as mediators of apoptosis. Since the recognition that CED-3,a protein required for developmental cell death, has sequence identitywith the mammalian cysteine protease interleukin-1 beta-convertingenzyme (ICE), a family of at least 10 related cysteine proteases hasbeen identified. These proteins are characterized by almost absolutespecificity for aspartic acid in the P1 position. All the caspases(ICE-like proteases) contain a conserved QACKG (where X is R, Z or G)pentapeptide active-site motif. Caspases are synthesized as inactiveproenzymes comprising an N-terminal peptide (Prodomain) together withone large and one small subunit. The crystal structures of bothcaspase-1 and caspase-3 show that the active enzyme is a heterotetramer,containing two small and two large subunits. Activation of caspasesduring apoptosis results in the cleavage of critical cellularsubstrates, including poly (ADP-riose) polymerase and lamins, soprecipitating the dramatic morphological changes of apoptosis (Cohen,1997, Biochem. J. 326:1-16). Therefore, it is also within the scope ofthe present invention to use a caspase as an apoptosis-inducing moiety.

Recently a few new proteins were cloned and identified as factorsrequired for mediating activity of proteins, mainly caspases, involvedin the apoptosis pathway. One factor was identified as the previouslyknown electron transfer protein, cytochrome c (Lin et al., 1996, Cell86:147-157), designed as Apaf-2. In addition to cytochrome c theactivation of caspase-3 requires two other cytosolic factors-Apaf-1 andApaf-3. Apaf-1 is a protein homologous to C. elegans CED-4, and Apaf-3was identified as a member of the caspase family, caspase-9. Bothfactors bind to each other via their respective NH2-terminal CED-3homologous domains, in the presence of cytochrome c, an event that leadsto caspase-9 activation. Activated caspase-9 in turn cleaves andactivates caspase-3 (Liu et al., 1996; Zou et al., 1997; Li et al.,1997). Another protein involved in the apoptotic pathway is DNAfragmentation factor (DFF), a heterodimer of 45 and 40 kd subunits thatfunctions downstream of caspase-3 to trigger fragmentation of genomicDNA into nucleosomal segments (Liu et al., 1997).

C. Chimeric Polypeptide Production

In accordance with the objects of the present invention, apolynucleotide that encodes a chimeric protein, mutant polypeptide,biologically active fragment of chimeric protein, or functionalequivalent thereof, may be used to generate recombinant DNA moleculesthat direct the expression of the chimeric protein, chimeric peptidefragments, or a functional equivalent thereof, in appropriate hostcells.

Due to the inherent degeneracy of the genetic code, other DNA sequencesthat encode substantially the same or a functionally equivalent aminoacid sequence, may be used in the practice of the invention of thecloning and expression of the chimeric protein. Such DNA sequencesinclude those capable of hybridizing to the chimeric sequences or theircomplementary sequences under stringent conditions. In one embodiment,the phrase “stringent conditions” as used herein refers to thosehybridizing conditions that (1) employ low ionic strength and hightemperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% SDS at 50° C.; (2) employ during hybridization a denaturingagent such as formamide, for example, 50% (vol/vol) formamide with a0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mMsodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrateat 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 MSodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC and 0.1% SDS.

Altered DNA sequences that may be used in accordance with the inventioninclude deletions, additions or substitutions of different nucleotideresidues resulting in a sequence that encodes the same or a functionallyequivalent fusion gene product. The gene product itself may containdeletions, additions or substitutions of amino acid residues within achimeric sequence, which result in a silent change thus producing afunctionally equivalent chimeric protein. Such amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine, histidine and arginine; amino acids with uncharged polar headgroups having similar hydrophilicity values include the following:glycine, asparagine, glutamine, serine, threonine, tyrosine; and aminoacids with nonpolar head groups include alanine, valine, isoleucine,leucine, phenylalanine, proline, methionine, tryptophan.

The DNA sequences of the invention may be engineered in order to alter achimeric coding sequence for a variety of ends, including but notlimited to, alterations which modify processing and expression of thegene product. For example, mutations may be introduced using techniqueswhich are well known in the art, e.g., site-directed mutagenesis, toinsert new restriction sites, to alter glycosylation patterns,phosphorylation, etc.

In an alternate embodiment of the invention, the coding sequence of thechimeric protein could be synthesized in whole or in part, usingchemical methods well known in the art. (See, for example, Caruthers etal., 1980; Crea and Horn, 1980; and Chow and Kempe, 1981). For example,active domains of the moieties can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography followed by chemical linkage to form achimeric protein. (e.g., see Creighton, 1983, Proteins Structures AndMolecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins, Structures and Molecular Principles, W.H.Freeman and Co., N.Y. pp. 34-49). Alternatively, the two moieties of thechimeric protein produced by synthetic or recombinant methods may beconjugated by chemical linkers according to methods well known in theart (Brinkmann and Pastan, 1994).

In order to express a biologically active chimeric protein, thenucleotide sequence coding for a chimeric protein, or a functionalequivalent, is inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence. The chimeric gene productsas well as host cells or cell lines transfected or transformed withrecombinant chimeric expression vectors can be used for a variety ofpurposes. These include but are not limited to generating antibodies(i.e., monoclonal or polyclonal) that bind to epitopes of the proteinsto facilitate their purification.

Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing the chimeric protein codingsequence and appropriate transcriptional/translational control signals.These methods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to expressthe chimeric protein coding sequence. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the chimeric protein coding sequence; yeast transformed withrecombinant yeast expression vectors containing the chimeric proteincoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the chimeric proteincoding sequence; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing the chimeric protein coding sequence; oranimal cell systems. It should be noted that since mostapoptosis-inducing proteins cause programmed cell death in mammaliancells, it is preferred that the chimeric protein of the invention beexpressed in prokaryotic or lower eukaryotic cells. Section 6illustrates that IL2-Bax may be efficiently expressed in E. coli.

The expression elements of each system vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter; cytomegalovirus promoter) and the like may be used;when cloning in insect cell systems, promoters such as the baculoviruspolyhedrin promoter may be used; when cloning in plant cell systems,promoters derived from the genome of plant cells (e.g., heat shockpromoters; the promoter for the small subunit of RUBISCO; the promoterfor the chlorophyll α/β binding protein) or from plant viruses (e.g.,the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may beused; when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter) may be used; when generating cell lines thatcontain multiple copies of the chimeric DNA, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the chimericprotein expressed. For example, when large quantities of chimericprotein are to be produced, vectors which direct the expression of highlevels of protein products that are readily purified may be desirable.Such vectors include but are not limited to the pHL906 vector (Fishmanet al., 1994), the E. coli expression vector pUR278 (Ruther et al.,1983), in which the chimeric protein coding sequence may be ligated intothe vector in frame with the lacZ coding region so that a hybrid AS-lacZprotein is produced; pIN vectors (Inouye and Inouye, 1989; Van Heeke andSchuster, 1989); and the like.

An alternative expression system which could be used to express chimericprotein is an insect system. In one such system, Autographa californicanuclear polyhidrosis virus (AcNPV) is used as a vector to expressforeign genes. The virus grows in Spodoptera frugiperda cells. Thechimeric protein coding sequence may be cloned into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).Successful insertion of the chimeric protein coding sequence will resultin inactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted gene isexpressed. (e.g., see Smith et al., 1983; U.S. Pat. No. 4,215,051).

Specific initiation signals may also be required for efficienttranslation of the inserted chimeric protein coding sequence. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where the entire chimeric gene, including its own initiation codonand adjacent sequences, is inserted into the appropriate expressionvector, no additional translational control signals may be needed.However, in cases where the chimeric protein coding sequence does notinclude its own initiation codon, exogenous translational controlsignals, including the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the chimeric protein coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. The presence of consensusN-glycosylation sites in a chimeric protein may require propermodification for optimal chimeric protein function. Different host cellshave characteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the chimeric protein. To this end, eukaryotic host cells whichpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the chimeric proteinmay be used. Such mammalian host cells include but are not limited toCHO, VERO, BHK, HeLa, COS, MDCK, 293, W138, and the like.

For long-term, high-yield production of recombinant chimeric proteins,stable expression is preferred. For example, cell lines which stablyexpress the chimeric protein may be engineered. Rather than usingexpression vectors which contain viral originals of replication, hostcells can be transformed with a chimeric coding sequence controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977),hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski,1962), and adenine phosphoribosyltransferase (Lowy et al., 1980) genescan be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980;O'Hare et al., 1981); gpt, which confers resistance to mycophenolic acid(Mulligan and Berg, 1981); neo, which confers resistance to theaminoglycoside G-418 (Colbere-Garapin et al., 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984) genes.Additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman andMulligan, 1988); and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L, 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

D. Protein Purification

The chimeric proteins of the invention can be purified by art-knowntechniques such as high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, affinity chromatography and thelike. The actual conditions used to purify a particular protein willdepend, in part, on factors such as net charge, hydrophobicity,hydrophilicity, etc., and will be apparent to those having skill in theart.

For affinity chromatography purification, any antibody whichspecifically binds the protein may be used. For the production ofantibodies, various host animals, including but not limited to rabbits,mice, rats, etc., may be immunized by injection with a chimeric proteinor a fragment thereof. The protein may be attached to a suitablecarrier, such as bovine serum albumin (BSA), by means of a side chainfunctional group or linkers attached to a side chain functional group.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhold limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmetter-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to a chimeric protein may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein(1975), the human B-cell hybridoma technique, (Kosbor et al., 1983; Coteet al., 1983) and the EBV-hybridoma technique (Cole et al., 1985). Inaddition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984; Neuberger et al., 1984; Takeda etal., 1985) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used.Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce chimericprotein-specific single chain antibodies for chimeric proteinpurification and detection.

V. Uses of Chimeric Polypeptide

Once a chimeric protein is expressed and purified, its identity andfunctional activities can be readily determined by methods well known inthe art. For example, antibodies to the two moieties of the protein maybe used to identify the protein in Western blot analysis. In addition,the chimeric protein can be tested for specific binding to target cellsin binding assays using a fluorescent-labeled or radiolabelled secondaryantibody.

A. In Vitro and Ex Vivo Uses

The chimeric polypeptides of the invention are useful for targetingspecific cell types in a cell mixture, and eliminating the target cellsby inducing apoptosis. The chimeric polypeptides of the invention arealso useful as a diagnostic reagent. The binding of a chimeric proteinto a target cell can be readily detected by using a secondary antibodyspecific for the apoptosis-inducing moiety. In that connection, thesecondary antibody or the chimeric protein enzyme or a radioisotope tofacilitate the detection of binding of the chimeric protein to a cell.

B. In Vivo Uses

In some embodiments, an effective amount of the chimeric polypeptides ofthe present invention are administered to a cell. In other embodiments,a therapeutically effective amount of the chimeric polypeptides of thepresent invention are administered to an individual for the treatment ofdisease. The term “effective amount” as used herein is defined as theamount of the chimeric polypeptides of the present invention which arenecessary to result in a physiological change in the cell or tissue towhich it is administered. The term “therapeutically effective amount” asused herein is defined as the amount of the chimeric polypeptides of thepresent invention that eliminate, decrease, delay, or minimize adverseeffects of a disease, such as cancer. A skilled artisan readilyrecognizes that in many cases the chimeric polypeptide may not provide acure but may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount ofchimeric polypeptide that provides a physiological change is consideredan “effective amount” or a “therapeutically effective amount.”

The chimeric proteins of the invention may be administered to a subjectper se or in the form of a pharmaceutical composition for the treatmentof cancer, autoimmunity, transplantation rejection, post-traumaticimmune responses and infectious diseases by targeting viral antigens,such as gp120 of HIV. More specifically, the chimeric polypeptides maybe useful in eliminating cells involved in immune cell-mediateddisorder, including lymphoma; autoimmunity, transplantation rejection,graft-versus-host disease, ischemia and stroke. Pharmaceuticalcompositions comprising the proteins of the invention may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the proteins into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For topical administration the proteins of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, inhalation, oral or pulmonary administration.

For injection, the proteins of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, the proteins may be in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the proteins can be readily formulated bycombining the proteins with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the proteins of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. For oral solid formulations such as, for example,powders, capsules and tablets, suitable excipients include fillers suchas sugars, e.g. lactose, sucrose, mannitol and sorbitol; cellulosepreparations such as maize starch, wheat starch, rice starch, potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the proteins may take the form of tablets,lozenges, etc. formulated in conventional manner.

For administration by inhalation, the proteins for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the protein and a suitable powder base suchas lactose or starch.

The proteins may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the proteins mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theproteins may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver proteins of the invention. Certain organicsolvents such as dimethylsulfoxide also may be employed, althoughusually at the cost of greater toxicity. Additionally, the proteins maybe delivered using a sustained-release system, such as semipermeablematrices of solid polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the proteins for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the chimeric protein, additional strategies for protein stabilizationmay be employed.

As the proteins of the invention may contain charged side chains ortermini, they may be included in any of the above-described formulationsas the free acids or bases or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts which substantiallyretain the biologic activity of the free bases and which are prepared byreaction with inorganic acids. Pharmaceutical salts tend to be moresoluble in aqueous and other protic solvents than are the correspondingfree base forms.

1. Effective Dosages

The proteins of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the proteins of the invention, or pharmaceuticalcompositions thereof, are administered or applied in a therapeuticallyeffective amount. A therapeutically effective amount is an amounteffective to ameliorate or prevent the symptoms, or prolong the survivalof, the patient being treated. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the proteins which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the proteins may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of protein administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs. In the case of autoimmune disorders,the drugs that may be used in combination with IL2-Bax of the inventioninclude, but are not limited to, steroid and non-steroidanti-inflammatory agents.

2. Toxicity

Preferably, a therapeutically effective dose of the chimeric proteinsdescribed herein will provide therapeutic benefit without causingsubstantial toxicity.

Toxicity of the proteins described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Proteinswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,Ch. 1, p. 1).

VI. Biological Functional Equivalents

As modifications and/or changes may be made in the structure of thepolynucleotides encoding the chimeric polypeptides of the presentinvention and/or the chimeric polypeptides themselves according to thepresent invention, while obtaining molecules having similar or improvedcharacteristics, such biologically functional equivalents are alsoencompassed within the present invention.

A. Modified Polynucleotides and Polypeptides

The biological functional equivalent may comprise a polynucleotide thathas been engineered to contain distinct sequences while at the same timeretaining the capacity to encode the “wild-type” or standard protein.This can be accomplished to the degeneracy of the genetic code, i.e.,the presence of multiple codons, which encode for the same amino acids.In one example, one of skill in the art may wish to introduce arestriction enzyme recognition sequence into a polynucleotide while notdisturbing the ability of that polynucleotide to encode a protein.

In another example, a polynucleotide encoding the chimeric polypeptidemay be (and may encode) a biological functional equivalent with moresignificant changes. Certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies, binding sites on substratemolecules, receptors, and the like. So-called “conservative” changes donot disrupt the biological activity of the polypeptide, as thestructural change is not one that impinges of the polypeptide's abilityto carry out its designed function. It is thus contemplated by theinventors that various changes may be made in the sequence of genes andproteins disclosed herein, while still fulfilling the goals of thepresent invention.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity. Biologically functionalequivalents are thus defined herein as those polypeptides (andpolynucleotides) in selected amino acids (or codons) may be substituted.Functional activity comprises the ability to kill a target cell for thesignal transduction pathway factor moiety or the ability to target acell specifically for the cell-specific targeting moiety.

In general, the shorter the length of the molecule, the fewer changesthat can be made within the molecule while retaining function. Longerdomains may have an intermediate number of changes. The full-lengthprotein will have the most tolerance for a larger number of changes.However, it must be appreciated that certain molecules or domains thatare highly dependent upon their structure may tolerate little or nomodification.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine and/or histidine are allpositively charged residues; that alanine, glycine and/or serine are alla similar size; and/or that phenylalanine, tryptophan and/or tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and/or histidine; alanine, glycineand/or serine; and/or phenylalanine, tryptophan and/or tyrosine; aredefined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and/or arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biological functional equivalent protein and/orpeptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and/or antigenicity, i.e., with a biological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and/or thosewithin ±0.5 are even more particularly preferred.

B. Altered Amino Acids

The present invention, in many aspects, relies on the synthesis ofpeptides and polypeptides in cyto, via transcription and translation ofappropriate polynucleotides. These peptides and polypeptides willinclude the twenty “natural” amino acids, and post-translationalmodifications thereof. However, in vitro peptide synthesis permits theuse of modified and/or unusual amino acids. A table of exemplary, butnot limiting, modified and/or unusual amino acids is provided hereinbelow. TABLE 2 Modified and/or Unusual Amino Acids Abbr. Amino AcidAbbr. Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad3-Aminoadipic acid Hyl Hydroxylysine BAla beta-alanine, beta-Amino- AHylallo-Hydroxylysine propionic acid Abu 2-Aminobutyric acid 3Hyp3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyprolinepiperidinic acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe2-Aminoheptanoic acid Aile allo-Isoleucine Aib 2-Aminoisobutyric acidMeGly N-Methylglycine, sarcosine BAib 3-Aminoisobutyric acid MeIleN-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva NorvalineDpm 2,2′-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionicacid Orn Ornithine EtGly N-Ethylglycine

C. Mimetics

In addition to the biological functional equivalents discussed above,the present inventors also contemplate that structurally similarcompounds may be formulated to mimic the key portions of peptide orpolypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins. Vita et al. (1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. Weisshoff et al. (1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn inducing properties.

Methods for generating specific structures have been disclosed in theart. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures renderthe peptide or protein more thermally stable, also increase resistanceto proteolytic degradation. Six, seven, eleven, twelve, thirteen andfourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

D. Liposome Targeting

Association of the chimeric polypeptide with a liposome may improvebiodistribution and other properties of the chimeric polypeptide. Forexample, liposome-mediated nucleic acid delivery and expression offoreign DNA in vitro has been very successful (Nicolau and Sene, 1982;Fraley et al., 1979; Nicolau et al., 1987). The feasibility ofliposome-mediated delivery and expression of foreign DNA in culturedchick embryo, HeLa and hepatoma cells has also been demonstrated (Wonget al., 1980). Successful liposome-mediated gene transfer in rats afterintravenous injection has also been accomplished (Nicolau et al., 1987).

It is contemplated that a liposome/chimeric polypeptide composition maycomprise additional materials for delivery to a tissue. For example, incertain embodiments of the invention, the lipid or liposome may beassociated with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In another example, thelipid or liposome may be complexed or employed in conjunction withnuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). Inyet further embodiments, the lipid may be complexed or employed inconjunction with both HVJ and HMG-1.

Targeted delivery is achieved by the addition of ligands withoutcompromising the ability of these liposomes deliver large amounts ofchimeric polypeptide. It is contemplated that this will enable deliveryto specific cells, tissues and organs. The targeting specificity of theligand-based delivery systems are based on the distribution of theligand receptors on different cell types. The targeting ligand mayeither be non-covalently or covalently associated with the lipidcomplex, and can be conjugated to the liposomes by a variety of methods.

E. Cross-Linkers

Bifunctional cross-linking reagents have been extensively used for avariety of purposes including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies.Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

Exemplary methods for cross-linking ligands to liposomes are describedin U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511, eachspecifically incorporated herein by reference in its entirety). Variousligands can be covalently bound to liposomal surfaces through thecross-linking of amine residues. Liposomes, in particular, multilamellarvesicles (MLV) or unilamellar vesicles such as microemulsified liposomes(MEL) and large unilamellar liposomes (LUVET), each containingphosphatidylethanolamine (PE), have been prepared by establishedprocedures. The inclusion of PE in the liposome provides an activefunctional residue, a primary amine, on the liposomal surface forcross-linking purposes. Ligands such as epidermal growth factor (EGF)have been successfully linked with PE-liposomes. Ligands are boundcovalently to discrete sites on the liposome surfaces. The number andsurface density of these sites will be dictated by the liposomeformulation and the liposome type. The liposomal surfaces may also havesites for non-covalent association. To form covalent conjugates ofligands and liposomes, cross-linking reagents have been studied foreffectiveness and biocompatibility. Cross-linking reagents includeglutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycoldiglycidyl ether (EGDE), and a water soluble carbodiimide, preferably1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Through the complexchemistry of cross-linking, linkage of the amine residues of therecognizing substance and liposomes is established.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S. Pat. No.5,889,155, specifically incorporated herein by reference in itsentirety). The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides and sugars. Table 3 details certainhetero-bifunctional cross-linkers considered useful in the presentinvention. TABLE 3 HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm ReactiveLength\after cross- Linker Toward Advantages and Applications linkingSMPT Primary amines Greater stability 11.2 A Sulfhydryls SPDP Primaryamines Thiolation 6.8 A Sulfhydryls Cleavable cross-linking LC-SPDPPrimary amines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC- Primaryamines Extended spacer arm 15.6 A SPDP Sulfhydryls Water-soluble SMCCPrimary amines Stable maleimide reactive group 11.6 A SulfhydrylsEnzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo-Primary amines Stable maleimide reactive group 11.6 A SMCC SulfhydrylsWater-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody conjugation 9.9 A Sulfhydryls Hapten-carrier proteinconjugation Sulfo- Primary amines Water-soluble 9.9 A MBS SulfhydrylsSIAB Primary amines Enzyme-antibody conjugation 10.6 A SulfhydrylsSulfo- Primary amines Water-soluble 10.6 A SIAB Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo- Primary amines Extended spacer arm 14.5 A SMPBSulfhydryls Water-soluble EDC/Sulfo- Primary amines Hapten-Carrierconjugation 0 NHS Carboxyl groups ABH Carbohydrates Reacts with sugargroups 11.9 A Nonselective

In instances where a particular polypeptide does not contain a residueamenable for a given cross-linking reagent in its native sequence,conservative genetic or synthetic amino acid changes in the primarysequence can be utilized.

VII. Combination Treatments/Cancer Therapies

In order to increase the effectiveness of a chimeric polypeptide of thepresent invention, or expression construct coding therefor, it may bedesirable to combine these compositions with other agents effective inthe treatment of hyperproliferative disease, such as anti-cancer agents.A hyperproliferative disease includes diseases and conditions that areassociated with any sort of abnormal cell growth or abnormal growthregulation. In methods of the present invention, preferably the patientis a human. A variety of hyperproliferative diseases can be treatedaccording to the methods of the present invention. Some of thehyperproliferative diseases contemplated for treatment in the presentinvention are psoriasis, rheumatoid arthritis (RA), inflammatory boweldisease (IBD), osteoarthritis (OA) and pre-neoplastic lesions in themouth, prostate, breast, lung etc. The present invention has importantramifications particularly with respect to one hyperproliferativedisease: cancer.

Thus, in certain embodiments, the hyperproliferative disease is furtherdefined as cancer. In still further embodiments, the cancer is melanoma,non-small cell lung, small-cell lung, lung, hepatocarcinoma,retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia,neuroblastoma, head, neck, breast, pancreatic, prostate, renal, bone,testicular, ovarian, mesothelioma, cervical, gastrointestinal, lymphoma,brain, colon, sarcoma or bladder. The cancer may include a tumorcomprised of tumor cells. In other embodiments, the hyperproliferativedisease is rheumatoid arthritis, inflammatory bowel disease,osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas,vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions(such as adenomatous hyperplasia and prostatic intraepithelialneoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

An “anti-cancer” agent is capable of negatively affecting cancer in asubject, for example, by killing cancer cells, inducing apoptosis incancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theexpression construct and the agent(s) or multiple factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir (Culver, et al., 1992). In the context ofthe present invention, it is contemplated that chimeric polypepidescould be used similarly in conjunction with chemotherapeutic,radiotherapeutic, gene therapy, or immunotherapeutic intervention, inaddition to other pro-apoptotic or cell cycle regulating agents.

Alternatively, the therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand expression construct would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone may contact the cell with both modalities within about 12-24 h ofeach other and, more preferably, within about 6-12 h of each other. Insome situations, it may be desirable to extend the time period fortreatment significantly, however, where several d (2, 3, 4, 5, 6 or 7)to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Various combinations may be employed, gene therapy is “A” and thesecondary agent, such as radio- or chemotherapy, is “B”: A/B/A B/A/BB/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the therapeutic expression constructs of the presentinvention to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described hyperproliferative cell therapy.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with gene therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

D. Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as a chimeric polypeptide of the present invention.Delivery of a chimeric polypeptide in conjuction with a second vectorencoding one of the following gene products will have a combinedanti-hyperproliferative effect on target tissues. Alternatively, asingle vector encoding both genes may be used. A variety of proteins areencompassed within the invention, some of which are described below.

1. Inducers of Cellular Proliferation

The proteins that induce cellular proliferation further fall intovarious categories dependent on function. The commonality of all ofthese proteins is their ability to regulate cellular proliferation. Forexample, a form of PDGF, the sis oncogene, is a secreted growth factor.Oncogenes rarely arise from genes encoding growth factors, and at thepresent, sis is the only known naturally-occurring oncogenic growthfactor. In one embodiment of the present invention, it is contemplatedthat anti-sense mRNA directed to a particular inducer of cellularproliferation is used to prevent expression of the inducer of cellularproliferation.

The proteins FMS, ErbA, ErbB and neu are growth factor receptors.Mutations to these receptors result in loss of regulatable function. Forexample, a point mutation affecting the transmembrane domain of the Neureceptor protein results in the neu oncogene. The erbA oncogene isderived from the intracellular receptor for thyroid hormone. Themodified oncogenic ErbA receptor is believed to compete with theendogenous thyroid hormone receptor, causing uncontrolled growth.

The largest class of oncogenes includes the signal transducing proteins(e.g., Src, Abl and Ras). The protein Src is a cytoplasmicprotein-tyrosine kinase, and its transformation from proto-oncogene tooncogene in some cases, results via mutations at tyrosine residue 527.In contrast, transformation of GTPase protein ras from proto-oncogene tooncogene, in one example, results from a valine to glycine mutation atamino acid 12 in the sequence, reducing ras GTPase activity.

The proteins Jun, Fos and Myc are proteins that directly exert theireffects on nuclear functions as transcription factors.

2. Inhibitors of Cellular Proliferation

The tumor suppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. The tumor suppressorsp53, p16 and C-CAM are described below.

High levels of mutant p53 have been found in many cells transformed bychemical carcinogenesis, ultraviolet radiation, and several viruses. Thep53 gene is a frequent target of mutational inactivation in a widevariety of human tumors and is already documented to be the mostfrequently mutated gene in common human cancers. It is mutated in over50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum ofother tumors.

The p53 gene encodes a 393-amino acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and areessential for the transforming ability of the oncogene. A single geneticchange prompted by point mutations can create carcinogenic p53. Unlikeother oncogenes, however, p53 point mutations are known to occur in atleast 30 distinct codons, often creating dominant alleles that produceshifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Another inhibitor of cellular proliferation is p16. The majortransitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16^(INK4) has been biochemically characterized as aprotein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p19, p21^(WAF1), and p27^(KIP1). Thep16^(INK4) gene maps to 9p21, a chromosome region frequently deleted inmany tumor types. Homozygous deletions and mutations of the p16^(INK4)gene are frequent in human tumor cell lines. This evidence suggests thatthe p16^(INK4) gene is a tumor suppressor gene. This interpretation hasbeen challenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

Other genes that may be employed according to the present inventioninclude Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL,MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions,anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu,raf, erb, fms, irk, ret, gsp, hst, abl, E1A, p300, genes involved inangiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or theirreceptors) and MCC.

3. Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto andCroce, 1986). The evolutionarily conserved Bcl-2 protein now isrecognized to be a member of a family of related proteins, which can becategorized as death agonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins whichshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or counteractBcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid,Bad, Harakiri).

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

F. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adehesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL would potentiate the apoptotic inducing abililties of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyerproliferative efficacy of the treatments. Inhibitors ofcell adehesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

VIII. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more chimeric polypeptides or chimericpolypeptides and at least one additional agent dissolved or dispersed ina pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains at least one chimeric polypeptide or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The chimeric polypeptide may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, inhalation (e.g. aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The chimeric polypeptide may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments, the chimeric polypeptide is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof, a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

IX. Lipid Compositions

In certain embodiments, the present invention employs a novelcomposition comprising one or more lipids associated with at least onechimeric polypeptide. A lipid is a substance that is characteristicallyinsoluble in water and extractable with an organic solvent. Lipidsinclude, for example, the substances comprising the fatty droplets thatnaturally occur in the cytoplasm as well as the class of compounds whichare well known to those of skill in the art which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes. Of course, compoundsother than those specifically described herein that are understood byone of skill in the art as lipids are also encompassed by thecompositions and methods of the present invention.

A lipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof.

A. Lipid Types

A neutral fat may comprise a glycerol and a fatty acid. A typicalglycerol is a three carbon alcohol. A fatty acid generally is a moleculecomprising a carbon chain with an acidic moeity (e.g., carboxylic acid)at an end of the chain. The carbon chain may of a fatty acid may be ofany length, however, it is preferred that the length of the carbon chainbe of from about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29, toabout 30 or more carbon atoms, and any range derivable therein. However,a preferred range is from about 14 to about 24 carbon atoms in the chainportion of the fatty acid, with about 16 to about 18 carbon atoms beingparticularly preferred in certain embodiments. In certain embodimentsthe fatty acid carbon chain may comprise an odd number of carbon atoms,however, an even number of carbon atoms in the chain may be preferred incertain embodiments. A fatty acid comprising only single bonds in itscarbon chain is called saturated, while a fatty acid comprising at leastone double bond in its chain is called unsaturated.

Specific fatty acids include, but are not limited to, linoleic acid,oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid,myristic acid, arachidic acid, palmitoleic acid, arachidonic acidricinoleic acid, tuberculosteric acid, lactobacillic acid. An acidicgroup of one or more fatty acids is covalently bonded to one or morehydroxyl groups of a glycerol. Thus, a monoglyceride comprises aglycerol and one fatty acid, a diglyceride comprises a glycerol and twofatty acids, and a triglyceride comprises a glycerol and three fattyacids.

A phospholipid generally comprises either glycerol or an sphingosinemoiety, an ionic phosphate group to produce an amphipathic compound, andone or more fatty acids. Types of phospholipids include, for example,phophoglycerides, wherein a phosphate group is linked to the firstcarbon of glycerol of a diglyceride, and sphingophospholipids (e.g.,sphingomyelin), wherein a phosphate group is esterified to a sphingosineamino alcohol. Another example of a sphingophospholipid is a sulfatide,which comprises an ionic sulfate group that makes the moleculeamphipathic. A phopholipid may, of course, comprise further chemicalgroups, such as for example, an alcohol attached to the phosphate group.Examples of such alcohol groups include serine, ethanolamine, choline,glycerol and inositol. Thus, specific phosphoglycerides include aphosphatidyl serine, a phosphatidyl ethanolamine, a phosphatidylcholine, a phosphatidyl glycerol or a phosphotidyl inositol. Otherphospholipids include a phosphatidic acid or a diacetyl phosphate. Inone aspect, a phosphatidylcholine comprises adioleoylphosphatidylcholine (a.k.a. cardiolipin), an eggphosphatidylcholine, a dipalmitoyl phosphalidycholine, a monomyristoylphosphatidylcholine, a monopalmitoyl phosphatidylcholine, a monostearoylphosphatidylcholine, a monooleoyl phosphatidylcholine, a dibutroylphosphatidylcholine, a divaleroyl phosphatidylcholine, a dicaproylphosphatidylcholine, a diheptanoyl phosphatidylcholine, a dicapryloylphosphatidylcholine or a distearoyl phosphatidylcholine.

A glycolipid is related to a sphinogophospholipid, but comprises acarbohydrate group rather than a phosphate group attached to a primaryhydroxyl group of the sphingosine. A type of glycolipid called acerebroside comprises one sugar group (e.g., a glucose or galactose)attached to the primary hydroxyl group. Another example of a glycolipidis a ganglioside (e.g., a monosialoganglioside, a GM1), which comprisesabout 2, about 3, about 4, about 5, about 6, to about 7 or so sugargroups, that may be in a branched chain, attached to the primaryhydroxyl group. In other embodiments, the glycolipid is a ceramide(e.g., lactosylceramide).

A steroid is a four-membered ring system derivative of a phenanthrene.Steroids often possess regulatory functions in cells, tissues andorganisms, and include, for example, hormones and related compounds inthe progestagen (e.g., progesterone), glucocoricoid (e.g., cortisol),mineralocorticoid (e.g., aldosterone), androgen (e.g., testosterone) andestrogen (e.g., estrone) families. Cholesterol is another example of asteroid, and generally serves structural rather than regulatoryfunctions. Vitamin D is another example of a sterol, and is involved incalcium absorption from the intestine.

A terpene is a lipid comprising one or more five carbon isoprene groups.Terpenes have various biological functions, and include, for example,vitamin A, coenyzme Q and carotenoids (e.g., lycopene and β-carotene).

B. Charged and Neutral Lipid Compositions

In certain embodiments, a lipid component of a composition is unchargedor primarily uncharged. In one embodiment, a lipid component of acomposition comprises one or more neutral lipids. In another aspect, alipid component of a composition may be substantially free of anionicand cationic lipids, such as certain phospholipids (e.g., phosphatidylcholine) and cholesterol. In certain aspects, a lipid component of anuncharged or primarily uncharged lipid composition comprises about 95%,about 96%, about 97%, about 98%, about 99% or 100% lipids without acharge, substantially uncharged lipid(s), and/or a lipid mixture withequal numbers of positive and negative charges.

In other aspects, a lipid composition may be charged. For example,charged phospholipids may be used for preparing a lipid compositionaccording to the present invention and can carry a net positive chargeor a net negative charge. In a non-limiting example, diacetyl phosphatecan be employed to confer a negative charge on the lipid composition,and stearylamine can be used to confer a positive charge on the lipidcomposition.

C. Making Lipids

Lipids can be obtained from natural sources, commercial sources orchemically synthesized, as would be known to one of ordinary skill inthe art. For example, phospholipids can be from natural sources, such asegg or soybean phosphatidylcholine, brain phosphatidic acid, brain orplant phosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine. In another example, lipids suitable for useaccording to the present invention can be obtained from commercialsources. For example, dimyristyl phosphatidylcholine (“DMPC”) can beobtained from Sigma Chemical Co., dicetyl phosphate (“DCP”) is obtainedfrom K & K Laboratories (Plainview, N.Y.); cholesterol (“Chol”) isobtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol(“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc.(Birmingham, Ala.). In certain embodiments, stock solutions of lipids inchloroform or chloroform/methanol can be stored at about −20° C.Preferably, chloroform is used as the only solvent since it is morereadily evaporated than methanol.

D. Lipid Composition Structures

In a preferred embodiment of the invention, the chimeric polypeptide maybe associated with a lipid. A chimeric polypeptide associated with alipid may be dispersed in a solution containing a lipid, dissolved witha lipid, emulsified with a lipid, mixed with a lipid, combined with alipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure. A lipid or lipid/chimericpolypeptide associated composition of the present invention is notlimited to any particular structure. For example, they may also simplybe interspersed in a solution, possibly forming aggregates which are notuniform in either size or shape. In another example, they may be presentin a bilayer structure, as micelles, or with a “collapsed” structure. Inanother non-limiting example, a lipofectamine (Gibco BRL)-chimericpolypeptide or Superfect (Qiagen)-chimeric polypeptide complex is alsocontemplated.

In certain embodiments, a lipid composition may comprise about 1%, about2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,or any range derivable therein, of a particular lipid, lipid type ornon-lipid component such as a drug, protein, sugar, nucleic acids orother material disclosed herein or as would be known to one of skill inthe art. In a non-limiting example, a lipid composition may compriseabout 10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. In another non-limiting example,a liposome may comprise about 4% to about 12% terpenes, wherein about 1%of the micelle is specifically lycopene, leaving about 3% to about 11%of the liposome as comprising other terpenes; and about 10% to about 35%phosphatidyl choline, and about 1% of a drug. Thus, it is contemplatedthat lipid compositions of the present invention may comprise any of thelipids, lipid types or other components in any combination or percentagerange.

1. Emulsions

A lipid may be comprised in an emulsion. A lipid emulsion is asubstantially permanent heterogenous liquid mixture of two or moreliquids that do not normally dissolve in each other, by mechanicalagitation or by small amounts of additional substances known asemulsifiers. Methods for preparing lipid emulsions and adding additionalcomponents are well known in the art (e.g., Modern Pharmaceutics, 1990,incorporated herein by reference).

For example, one or more lipids are added to ethanol or chloroform orany other suitable organic solvent and agitated by hand or mechanicaltechniques. The solvent is then evaporated from the mixture leaving adried glaze of lipid. The lipids are resuspended in aqueous media, suchas phosphate buffered saline, resulting in an emulsion. To achieve amore homogeneous size distribution of the emulsified lipids, the mixturemay be sonicated using conventional sonication techniques, furtheremulsified using microfluidization (using, for example, aMicrofluidizer, Newton, Mass.), and/or extruded under high pressure(such as, for example, 600 psi) using an Extruder Device (LipexBiomembranes, Vancouver, Canada).

2. Micelles

A lipid may be comprised in a micelle. A micelle is a cluster oraggregate of lipid compounds, generally in the form of a lipidmonolayer, and may be prepared using any micelle producing protocolknown to those of skill in the art (e.g., Canfield et al., 1990;El-Gorab et al, 1973; Colloidal Surfactant, 1963; and Catalysis inMicellar and Macromolecular Systems, 1975, each incorporated herein byreference). For example, one or more lipids are typically made into asuspension in an organic solvent, the solvent is evaporated, the lipidis resuspended in an aqueous medium, sonicated and then centrifuged.

3. Liposomes

In particular embodiments, a lipid comprises a liposome. A “liposome” isa generic term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes may be characterized as having vesicularstructures with a bilayer membrane, generally comprising a phospholipid,and an inner medium that generally comprises an aqueous composition.

A multilamellar liposome has multiple lipid layers separated by aqueousmedium. They form spontaneously when lipids comprising phospholipids aresuspended in an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Lipophilic molecules or molecules with lipophilicregions may also dissolve in or associate with the lipid bilayer.

In certain less preferred embodiments, phospholipids from naturalsources, such as egg or soybean phosphatidylcholine, brain phosphatidicacid, brain or plant phosphatidylinositol, heart cardiolipin and plantor bacterial phosphatidylethanolamine are preferably not used as theprimary phosphatide, i.e., constituting 50% or more of the totalphosphatide composition or a liposome, because of the instability andleakiness of the resulting liposomes.

In particular embodiments, a lipid and/or chimeric polypeptide may be,for example, encapsulated in the aqueous interior of a liposome,interspersed within the lipid bilayer of a liposome, attached to aliposome via a linking molecule that is associated with both theliposome and the chimeric polypeptide, entrapped in a liposome,complexed with a liposome, etc.

a. Making Liposomes

A liposome used according to the present invention can be made bydifferent methods, as would be known to one of ordinary skill in theart. Phospholipids can form a variety of structures other than liposomeswhen dispersed in water, depending on the molar ratio of lipid to water.At low ratios the liposome is the preferred structure.

For example, a phospholipid (Avanti Polar Lipids, Alabaster, Ala.), suchas for example the neutral phospholipid dioleoylphosphatidylcholine(DOPC), is dissolved in tert-butanol. The lipid(s) is then mixed withthe chimeric polypeptide, and/or other component(s). Tween 20 is addedto the lipid mixture such that Tween 20 is about 5% of the composition'sweight. Excess tert-butanol is added to this mixture such that thevolume of tert-butanol is at least 95%. The mixture is vortexed, frozenin a dry ice/acetone bath and lyophilized overnight. The lyophilizedpreparation is stored at −20° C. and can be used up to three months.When required the lyophilized liposomes are reconstituted in 0.9%saline. The average diameter of the particles obtained using Tween 20for encapsulating the chimeric polypeptide is about 0.7 to about 1.0 μmin diameter.

Alternatively, a liposome can be prepared by mixing lipids in a solventin a container, e.g., a glass, pear-shaped flask. The container shouldhave a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film isresuspended. The aqueous liposomes can be then separated into aliquots,each placed in a vial, lyophilized and sealed under vacuum.

In other alternative methods, liposomes can be prepared in accordancewith other known laboratory procedures (e.g., see Bangham et al., 1965;Gregoriadis, 1979; Deamer and Uster 1983, Szoka and Papahadjopoulos,1978, each incorporated herein by reference in relevant part). Thesemethods differ in their respective abilities to entrap aqueous materialand their respective aqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as described abovemay be dehydrated and reconstituted in a solution of inhibitory peptideand diluted to an appropriate concentration with an suitable solvent,e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer.Unencapsulated additional materials, such as agents including but notlimited to hormones, drugs, nucleic acid constructs and the like, areremoved by centrifugation at 29,000×g and the liposomal pellets washed.The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount ofadditional material or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amount ofadditional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriate concentrationsand stored at 4° C. until use. A pharmaceutical composition comprisingthe liposomes will usually include a sterile, pharmaceuticallyacceptable carrier or diluent, such as water or saline solution.

The size of a liposome varies depending on the method of synthesis.Liposomes in the present invention can be a variety of sizes. In certainembodiments, the liposomes are small, e.g., less than about 100 nm,about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less than about50 nm in external diameter. In preparing such liposomes, any protocoldescribed herein, or as would be known to one of ordinary skill in theart may be used. Additional non-limiting examples of preparing liposomesare described in U.S. Pat. Nos. 4,728,578, 4,728,575, 4,737,323,4,533,254, 4,162,282, 4,310,505, and 4,921,706; InternationalApplications PCT/US85/01161 and PCT/US89/05040; U.K. Patent ApplicationGB 2193095 A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al. 1987;Mayhew et al., 1984; Cheng et al., 1987; and Liposome Technology, 1984,each incorporated herein by reference).

A liposome suspended in an aqueous solution is generally in the shape ofa spherical vesicle, having one or more concentric layers of lipidbilayer molecules. Each layer consists of a parallel array of moleculesrepresented by the formula XY, wherein X is a hydrophilic moiety and Yis a hydrophobic moiety. In aqueous suspension, the concentric layersare arranged such that the hydrophilic moieties tend to remain incontact with an aqueous phase and the hydrophobic regions tend toself-associate. For example, when aqueous phases are present both withinand without the liposome, the lipid molecules may form a bilayer, knownas a lamella, of the arrangement XY-YX. Aggregates of lipids may formwhen the hydrophilic and hydrophobic parts of more than one lipidmolecule become associated with each other. The size and shape of theseaggregates will depend upon many different variables, such as the natureof the solvent and the presence of other compounds in the solution.

The production of lipid formulations often is accomplished by sonicationor serial extrusion of liposomal mixtures after (I) reverse phaseevaporation (II) dehydration-rehydration (III) detergent dialysis and(IV) thin film hydration. In one aspect, a contemplated method forpreparing liposomes in certain embodiments is heating sonicating, andsequential extrusion of the lipids through filters or membranes ofdecreasing pore size, thereby resulting in the formation of small,stable liposome structures. This preparation produces liposomal/chimericpolypeptide or liposomes only of appropriate and uniform size, which arestructurally stable and produce maximal activity. Such techniques arewell-known to those of skill in the art (see, for example Lang et al.,1990).

Once manufactured, lipid structures can be used to encapsulate compoundsthat are toxic (e.g., chemotherapeutics) or labile (e.g., nucleic acids)when in circulation. The physical characteristics of liposomes depend onpH, ionic strength and/or the presence of divalent cations. Liposomescan show low permeability to ionic and/or polar substances, but atelevated temperatures undergo a phase transition which markedly alterstheir permeability. The phase transition involves a change from aclosely packed, ordered structure, known as the gel state, to a looselypacked, less-ordered structure, known as the fluid state. This occurs ata characteristic phase-transition temperature and/or results in anincrease in permeability to ions, sugars and/or drugs. Liposomalencapsulation has resulted in a lower toxicity and a longer serumhalf-life for such compounds (Gabizon et al, 1990).

Liposomes interact with cells to deliver agents via four differentmechanisms: Endocytosis by phagocytic cells of the reticuloendothelialsystem such as macrophages and/or neutrophils; adsorption to the cellsurface, either by nonspecific weak hydrophobic and/or electrostaticforces, and/or by specific interactions with cell-surface components;fusion with the plasma cell membrane by insertion of the lipid bilayerof the liposome into the plasma membrane, with simultaneous release ofliposomal contents into the cytoplasm; and/or by transfer of liposomallipids to cellular and/or subcellular membranes, and/or vice versa,without any association of the liposome contents. Varying the liposomeformulation can alter which mechanism is operative, although more thanone may operate at the same time.

Numerous disease treatments are using lipid based gene transferstrategies to enhance conventional or establish novel therapies, inparticular therapies for treating hyperproliferative diseases. Advancesin liposome formulations have improved the efficiency of gene transferin vivo (Templeton et al., 1997) and it is contemplated that liposomesare prepared by these methods. Alternate methods of preparinglipid-based formulations for nucleic acid delivery are described (WO99/18933).

In another liposome formulation, an amphipathic vehicle called a solventdilution microcarrier (SDMC) enables integration of particular moleculesinto the bi-layer of the lipid vehicle (U.S. Pat. No. 5,879,703). TheSDMCs can be used to deliver lipopolysaccharides, polypeptides, nucleicacids and the like. Of course, any other methods of liposome preparationcan be used by the skilled artisan to obtain a desired liposomeformulation in the present invention.

b. Targeting Ligands

The targeting ligand can be either anchored in the hydrophobic portionof the complex or attached to reactive terminal groups of thehydrophilic portion of the complex. The targeting ligand can be attachedto the liposome via a linkage to a reactive group, e.g., on the distalend of the hydrophilic polymer. Preferred reactive groups include aminogroups, carboxylic groups, hydrazide groups, and thiol groups. Thecoupling of the targeting ligand to the hydrophilic polymer can beperformed by standard methods of organic chemistry that are known tothose skilled in the art. In certain embodiments, the totalconcentration of the targeting ligand can be from about 0.01 to about10% mol.

Targeting ligands are any ligand specific for a characteristic componentof the targeted region. Preferred targeting ligands include proteinssuch as polyclonal or monoclonal antibodies, antibody fragments, orchimeric antibodies, enzymes, or hormones, or sugars such as mono-,oligo- and poly-saccharides (see, Heath et al., Chem. Phys. Lipids40:347 (1986)) For example, disialoganglioside GD2 is a tumor antigenthat has been identified neuroectodermal origin tumors, such asneuroblastoma, melanoma, small-cell lung carcinoma, glioma and certainsarcomas (Mujoo et al., 1986, Schulz et al., 1984). Liposomes containinganti-disialoganglioside GD2 monoclonal antibodies have been used to aidthe targeting of the liposomes to cells expressing the tumor antigen(Montaldo et al., 1999; Pagan et al., 1999). In another non-limitingexample, breast and gynecological cancer antigen specific antibodies aredescribed in U.S. Pat. No. 5,939,277, incorporated herein by reference.In a further non-limiting example, prostate cancer specific antibodiesare disclosed in U.S. Pat. No. 6,107,090, incorporated herein byreference. Thus, it is contemplated that the antibodies described hereinor as would be known to one of ordinary skill in the art may be used totarget specific tissues and cell types in combination with thecompositions and methods of the present invention. In certainembodiments of the invention, contemplated targeting ligands interactwith integrins, proteoglycans, glycoproteins, receptors or transporters.Suitable ligands include any that are specific for cells of the targetorgan, or for structures of the target organ exposed to the circulationas a result of local pathology, such as tumors.

In certain embodiments of the present invention, in order to enhance thetransduction of cells, to increase transduction of target cells, or tolimit transduction of undesired cells, antibody or cyclic peptidetargeting moieties (ligands) are associated with the lipid complex. Suchmethods are known in the art. For example, liposomes have been describedfurther that specifically target cells of the mammalian central nervoussystem (U.S. Pat. No. 5,786,214, incorporated herein by reference). Theliposomes are composed essentially ofN-glutarylphosphatidylethanolamine, cholesterol and oleic acid, whereina monoclonal antibody specific for neuroglia is conjugated to theliposomes. It is contemplated that a monoclonal antibody or antibodyfragment may be used to target delivery to specific cells, tissues, ororgans in the animal, such as for example, brain, heart, lung, liver,etc.

Still further, a chimeric polypeptide may be delivered to a target cellvia receptor-mediated delivery and/or targeting vehicles comprising alipid or liposome. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis that will be occurringin a target cell. In view of the cell type-specific distribution ofvarious receptors, this delivery method adds another degree ofspecificity to the present invention.

Thus, in certain aspects of the present invention, a ligand will bechosen to correspond to a receptor specifically expressed on the targetcell population. A cell-specific chimeric polypeptide delivery and/ortargeting vehicle may comprise a specific binding ligand in combinationwith a liposome. The chimeric polypeptide to be delivered are housedwithin a liposome and the specific binding ligand is functionallyincorporated into a liposome membrane. The liposome will thusspecifically bind to the receptor(s) of a target cell and deliver thecontents to a cell. Such systems have been shown to be functional usingsystems in which, for example, epidermal growth factor (EGF) is used inthe receptor-mediated delivery of a nucleic acid to cells that exhibitupregulation of the EGF receptor.

In certain embodiments, a receptor-mediated delivery and/or targetingvehicles comprise a cell receptor-specific ligand and a chimericpolypeptide-binding agent. Others comprise a cell receptor-specificligand to which chimeric polypeptide to be delivered has beenoperatively attached. For example, several ligands have been used forreceptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990;Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. In another example, specific delivery inthe context of another mammalian cell type has been described (Wu andWu, 1993; incorporated herein by reference).

In still further embodiments, the specific binding ligand may compriseone or more lipids or glycoproteins that direct cell-specific binding.For example, lactosyl-ceramide, a galactose-terminal asialganglioside,have been incorporated into liposomes and observed an increase in theuptake of the insulin gene by hepatocytes (Nicolau et al., 1987). Theasialoglycoprotein, asialofetuin, which contains terminal galactosylresidues, also has been demonstrated to target liposomes to the liver(Spanjer and Scherphof, 1983; Hara et al., 1996). The sugars mannosyl,fucosyl or N-acetyl glucosamine, when coupled to the backbone of apolypeptide, bind the high affinity manose receptor (U.S. Pat. No.5,432,260, specifically incorporated herein by reference in itsentirety). It is contemplated that the cell or tissue-specifictransforming constructs of the present invention can be specificallydelivered into a target cell or tissue in a similar manner.

In another example, lactosyl ceramide, and peptides that target the LDLreceptor related proteins, such as apolipoprotein E3 (“Apo E”) have beenuseful in targeting liposomes to the liver (Spanjer and Scherphof, 1983;WO 98/0748).

Folate and the folate receptor have also been described as useful forcellular targeting (U.S. Pat. No. 5,871,727). In this example, thevitamin folate is coupled to the complex. The folate receptor has highaffinity for its ligand and is overexpressed on the surface of severalmalignant cell lines, including lung, breast and brain tumors.Anti-folate such as methotrexate may also be used as targeting ligands.Transferrin mediated delivery systems target a wide range of replicatingcells that express the transferrin receptor (Gilliland et al., 1980).

c. Liposome/Nucleic Acid Combinations

In certain embodiments, a liposome/chimeric polypeptide may comprise anucleic acid, such as, for example, an oligonucleotide, a polynucleotideor a nucleic acid construct (e.g., an expression vector). Where abacterial promoter is employed in the DNA construct that is to betransfected into eukaryotic cells, it also will be desirable to includewithin the liposome an appropriate bacterial polymerase.

It is contemplated that when the liposome/chimeric polypeptidecomposition comprises a cell or tissue specific nucleic acid, thistechnique may have applicability in the present invention. In certainembodiments, lipid-based non-viral formulations provide an alternativeto viral gene therapies. Although many cell culture studies havedocumented lipid-based non-viral gene transfer, systemic gene deliveryvia lipid-based formulations has been limited. A major limitation ofnon-viral lipid-based gene delivery is the toxicity of the cationiclipids that comprise the non-viral delivery vehicle. The in vivotoxicity of liposomes partially explains the discrepancy between invitro and in vivo gene transfer results. Another factor contributing tothis contradictory data is the difference in liposome stability in thepresence and absence of serum proteins. The interaction betweenliposomes and serum proteins has a dramatic impact on the stabilitycharacteristics of liposomes (Yang and Huang, 1997). Cationic liposomesattract and bind negatively charged serum proteins. Liposomes coated byserum proteins are either dissolved or taken up by macrophages leadingto their removal from circulation. Current in vivo liposomal deliverymethods use aerosolization, subcutaneous, intradermal, intratumoral, orintracranial injection to avoid the toxicity and stability problemsassociated with cationic lipids in the circulation. The interaction ofliposomes and plasma proteins is largely responsible for the disparitybetween the efficiency of in vitro (Felgner et al., 1987) and in vivogene transfer (Zhu et al., 1993; Philip et al., 1993; Solodin et al.,1995; Liu et al., 1995; Thierry et al., 1995; Tsukamoto et al., 1995;Aksentijevich et al., 1996).

An exemplary method for targeting viral particles to cells that lack asingle cell-specific marker has been described (U.S. Pat. No.5,849,718). In this method, for example, antibody A may have specificityfor tumor, but also for normal heart and lung tissue, while antibody Bhas specificity for tumor but also normal liver cells. The use ofantibody A or antibody B alone to deliver an anti-proliferative nucleicacid to the tumor would possibly result in unwanted damage to heart andlung or liver cells. However, antibody A and antibody B can be usedtogether for improved cell targeting. Thus, antibody A is coupled to agene encoding an anti-proliferative nucleic acid and is delivered, via areceptor mediated uptake system, to tumor as well as heart and lungtissue. However, the gene is not transcribed in these cells as they lacka necessary transcription factor. Antibody B is coupled to a universallyactive gene encoding the transcription factor necessary for thetranscription of the anti-proliferative nucleic acid and is delivered totumor and liver cells. Therefore, in heart and lung cells only theinactive anti-proliferative nucleic acid is delivered, where it is nottranscribed, leading to no adverse effects. In liver cells, the geneencoding the transcription factor is delivered and transcribed, but hasno effect because no an anti-proliferative nucleic acid gene is present.In tumor cells, however, both genes are delivered and the transcriptionfactor can activate transcription of the anti-proliferative nucleicacid, leading to tumor-specific toxic effects.

The addition of targeting ligands for gene delivery for the treatment ofhyperproliferative diseases permits the delivery of genes whose geneproducts are more toxic than do non-targeted systems. Examples of themore toxic genes that can be delivered includes pro-apoptotic genes suchas Bax and Bak plus genes derived from viruses and other pathogens suchas the adenoviral E4orf4 and the E. coli purine nucleosidephosphorylase, a so-called “suicide gene” which converts the prodrug6-methylpurine deoxyriboside to toxic purine 6-methylpurine. Otherexamples of suicide genes used with prodrug therapy are the E. colicytosine deaminase gene and the HSV thymidine kinase gene.

It is also possible to utilize untargeted or targeted lipid complexes togenerate recombinant or modified viruses in vivo. For example, two ormore plasmids could be used to introduce retroviral sequences plus atherapeutic gene into a hyperproliferative cell. Retroviral proteinsprovided in trans from one of the plasmids would permit packaging of thesecond, therapeutic gene-carrying plasmid. Transduced cells, therefore,would become a site for production of non-replicative retrovirusescarrying the therapeutic gene. These retroviruses would then be capableof infecting nearby cells. The promoter for the therapeutic gene may ormay not be inducible or tissue specific.

Similarly, the transferred nucleic acid may represent the DNA for areplication competent or conditionally replicating viral genome, such asan adenoviral genome that lacks all or part of the adenoviral E1a or E2bregion or that has one or more tissue-specific or inducible promotersdriving transcription from the E1a and/or E1b regions. This replicatingor conditional replicating nucleic acid may or may not contain anadditional therapeutic gene such as a tumor suppressor gene oranti-oncogene.

d. Lipid Administration

The actual dosage amount of a lipid composition (e.g., aliposome-chimeric polypeptide) administered to a patient can bedetermined by physical and physiological factors such as body weight,severity of condition, idiopathy of the patient and on the route ofadministration. With these considerations in mind, the dosage of a lipidcomposition for a particular subject and/or course of treatment canreadily be determined.

The present invention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, rectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intravesicularlly, mucosally, intrapericardially, orally, topically,locally and/or using aerosol, injection, infusion, continuous infusion,localized perfusion bathing target cells directly or via a catheterand/or lavage.

X. Antibody Preparation

A. Polyclonal Antibodies

Polyclonal antibodies are useful in the present invention regardingmultiple embodiments for the chimeric polypeptides. Polyclonalantibodies to the chimeric polypeptides generally are raised in animalsby multiple subcutaneous (sc) or intraperitoneal (ip) injections of thechimeric polypeptide and an adjuvant. It may be useful to conjugate thechimeric polypeptides or a fragment containing the target amino acidsequence to a protein that is immunogenic in the species to beimmunized, e.g. keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glytaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg of 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to 1/10 the original amount of conjugate inFreud's complete adjuvant by subcutaneous injection at multiple sites. 7to 14 days later the animals are bled and the serum is assayed foranti-chimeric polypeptides antibody titer. Animals are boosted until thetiter plateaus. Preferably, the animal boosted with the conjugate of thesame chimeric polypeptides, but conjugated to a different protein and/orthrough a different cross-linking reagent. Conjugates also can be madein recombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

B. Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the anti-chimeric polypeptide monoclonal antibodies of theinvention may be made using the hybridoma method first described byKohler and Milstein (1975), or may be made by recombinant DNA methods[Cabilly et al., U.S. Pat. No. 4,816,567].

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell [Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)].

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against chimericpolypeptides. Preferably, the binding specificity of monoclonalantibodies produced by hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson and Pollard (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-104(Academic Press, 1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences(Morrison et al., 1984), or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of ananti-chimeric polypeptide monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a chimericpolypeptide and another antigen-combining site having specificity for adifferent antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., (1962); David et al. (1974); Pain et al. (1981); andNygren (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a chimeric polypeptide or an immunologically reactiveportion thereof) to compete with the test sample analyte (chimericpolypeptides) for binding with a limited amount of antibody. The amountof chimeric polypeptides in the test sample is inversely proportional tothe amount of standard that becomes bound to the antibodies. Tofacilitate determining the amount of standard that becomes bound, theantibodies generally are insolubilized before or after the competition,so that the standard and analyte that are bound to the antibodies mayconveniently be separated from the standard and analyte which remainunbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex (U.S. Pat. No. 4,376,110). The second antibody may itselfbe labeled with a detectable moiety (direct sandwich assays) or may bemeasured using an anti-immunoglobulin antibody that is labeled with adetectable moiety (indirect sandwich assay). For example, one type ofsandwich assay is an ELISA assay, in which case the detectable moiety isan enzyme.

C. Humanized Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al. (1986); Riechmann et al. (1988); Verhoeyen et al. (1988)),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed Aug. 21, 1992,which is a continuation-in-part of application Ser. No. 07/715,272 filedJun. 14, 1991.

D. Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor(1984), and Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.(1993).

Alternatively, the phage display technology (McCafferty et al. (1990)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.

Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimicks some of theproperties of the B-cell. Phage display can be performed in a variety offormats; for their review see, e.g. Johnson and Chiswell (1993). Severalsources of V-gene segments can be used for phage display. Clackson etal. (1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al. (1991), or Griffith et al. (1993).In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (Marks et al., 1992).In this method, the affinity of “primary” human antibodies obtained byphage display can be improved by sequentially replacing the heavy andlight chain V region genes with repertoires of naturally occurringvariants (repertoires) of V domain genes obtained from unimmunizeddonors. This techniques allows the production of antibodies and antibodyfragments with affinities in the nM range. A strategy for making verylarge phage antibody repertoires (also known as “the mother-of-alllibraries”) has been described by Waterhouse et al. (1993), and theisolation of a high affinity human antibody directly from such largephage library is reported by Griffith et al. (1994). Gene shuffling canalso be used to derive human antibodies from rodent antibodies, wherethe human antibody has similar affinities and specificities to thestarting rodent antibody. According to this method, which is alsoreferred to as “epitope imprinting”, the heavy or light chain V domaingene of rodent antibodies obtained by phage display technique isreplaced with a repertoire of human V domain genes, creatingrodent-human chimeras. Selection on antigen results in isolation ofhuman variable capable of restoring a functional antigen-binding site,i.e. the epitope governs (imprints) the choice of partner. When theprocess is repeated in order to replace the remaining rodent V domain, ahuman antibody is obtained (see PCT patent application WO 93/06213,published Apr. 1, 1993). Unlike traditional humanization of rodentantibodies by CDR grafting, this technique provides completely humanantibodies, which have no framework or CDR residues of rodent origin.

E. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for achimeric polypeptide, the other one is for any other antigen, andpreferably for another receptor or receptor subunit. For example,bispecific antibodies specifically binding a chimeric polypeptide andneurotrophic factor, or two different chimeric polypeptides are withinthe scope of the present invention.

F. Methods for Making Bispecific Antibodies are Known in the Art

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello (1983)). Because of the random assortment of immunoglobulinheavy and light chains, these hybridomas (quadromas) produce a potentialmixture of 10 different antibody molecules, of which only one has thecorrect bispecific structure. The purification of the correct molecule,which is usually done by affinity chromatography steps, is rathercumbersome, and the product yields are low. Similar procedures aredisclosed in PCT application publication No. WO 93/08829 (published May13, 1993), and in Traunecker et al. (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH₂ and CH₃ regions. Itis preferred to have the first heavy chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are cotransfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed incopending application Ser. No. 07/931,811 filed Aug. 17, 1992.

For further details of generating bispecific antibodies see, forexample, Suresh et al. (1986).

G. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

EXAMPLES

The following is an illustration of preferred embodiments for practicingthe present invention. However, they are not limiting examples. Otherexamples and methods are possible in practicing the present invention.

Example 1 Materials

The following materials were utilized for multiple Examples describedherein. The PCR reagents were obtained from Fisher Scientific(Pittsburgh, Pa.), and the molecular biology enzymes were purchased fromBoehringer Mannheim (Indianapolis, Ind.) or New England Biolabs(Beverly, Mass.). Bacterial strains, pET bacterial expression plasmidsand recombinant enterokinase were obtained from Novagen (Madison, Wis.).All other chemicals were from Sigma Chemical Company (St. Louis, Mo.) orFisher Scientific (Pittsburg, Pa.). Metal affinity resin (Talon orNichel agrose) was obtained from Clontech Laboratories (Palo Alto,Calif.). Tissue culture reagents were from Gibco BRL (Gaithersburg,Md.).

Human cutaneous T cell lymphoma (Hut78) from American Type CultureCollection (ATCC, Manassas, Va.) cultured in RPMI 1640 medium containing10% fetal bovine serum (FBS). Porcine aortic endothelial cellstransfected with either flt-1 receptor (PAE-Flt-1) or the flk-1/KDRreceptor (PAE-Flk-1) were a gift from Dr. J. Waltenberger and culturedin F12 Nutrient Mixture (HAM) with 10% FBS. The human melanoma A375Mcell-line was obtained from Dr. I. J. Fidler of the University of TexasM D Anderson Cancer Center (Houston, Tex.) and were cultured in MEMsupplemented with 10% FBS.

Example 2 Methods—Cloning Human Granzyme B Gene

The following methods were utilized at least for Example 13. Hut78 RNAwas isolated using GlassMAX RNA Microisolation Spin Cartridge System(Gibco BRL), and the quanitity of total RNA was then determined. GenomicDNA was then removed by incubating the sample with DNase I for 15 min atroom temperature. The DNase I was inactivated by adding EDTA solutionheating for 15 min at 65° C. The SUPERSCRIPT First-Strand SynthesisSystem for RT-PCR (Gibco BRL) was used to synthesize the first-strandwith oligo (dT). The target cDNA (pre-mature human Granzyme B cDNA) wasamplified using the primers: NcoIgb (5′ to 3′):GGTGGCGGTGGCTCCATGGAACCAATCCTGCTTCTG (SEQ ID NO:1) and Cxholgb (5′ to3′): GCCACCGCCTCCCTCGAGCTATTAGTAGCGTTTCATGGT (SEQ ID NO:2) by PCR. PCRconditions included denaturation at 95° C. for 5 min, PCR cycle of 94°C. for 1 min, 50° C. for 1 min, 72° C. for 1 min, for a total of 30cycles; extension step was 72° C. for 5 min. A 1% agarose gel was run toconfirm the PCR product. The PCR product was then cloned into PCR 2.1 TAvector (Invitrogen; Carlsbad, Calif.) and designated gbTA. The gbTA wastransformed into INVαF′ competent cells, and the positive clones werescreened by blue/white colony screening or by PCR methods. The DNAs forpositive clones were isolated by using QIAprep Spin prep kit (Qiagen;Valencia, Calif.) and sequenced to confirm human granzyme B gene; thecorrect clone was identified as gbTA-2 (clone #2).

Example 3 Methods—Construction of Granzyme B-Vegf121 or GranzymeB-ScFvMEL Fusion Genes

The following fusion constructs were utilized in multiple Examplesdescribed herein. The fusion construct Granzyme B-vegf121 was anEk-Granzyme B-G4S linker-Vegf121 format. The construction was based onover-lap PCR method. Briefly, granzyme B coding sequence was amplifiedfrom gbTA-2 by PCR using the primers: NgbEK (5′ to 3′):GGTACCGACGACGACGACAAGATCATCGGGGGACATGAG, Cgb (5′ to 3′) (SEQ ID NO:3)and GGAGCCACCGCCACCGTAGCGTTTCATGGT (SEQ ID NO:4). These were designed todelete the signal sequence of pre-mature granzyme B and insert anenterokinase cleavage site at the N-terminus, in addition to adding aG4S linker sequence to the C-terminus in order to link to vegf121 gene.Vegf121 sequence was amplified from a plasmid pET22-vegf121 (a gift fromDr. Phil Thorpe's group, the University of Texas Southwest MedicalSchool, Dallas, Tex.) by PCR using primers: Nvegf (5′ to 3′)GGTGGCGGTGGCTCCGCACCCATGGCAGAA (SEQ ID NO:5) and CxhoI veg (5′ to 3′)AAGGCTCGTGTCGACCTCGAGTCATTACCGCCTCGGCTTGTC (SEQ ID NO:6). ScFvMELsequence was amplified from a plasmid pET32-scFvMEL/TNF by PCR usingprimers: Nzme2 (5′ to 3′)GGTGGCGGTGGCTCCACGGACATTGTGATGACCCAGTCTCAAAAATTC (SEQ ID NO:7) and Czme2(5′ to 3′) GGAGCCACCGCCACCCTCGAGCTATCATGAGGAGACGGTGAGAGTGGT (SEQ IDNO:8). These primers added G4S linker sequence to the N-terminus tooverlap PCR link to the C-terminus of granzyme B, and a Xho I site wasincorporated at the C-terminus to facilitate subsequent cloning steps.Two stop codons were added at the C-terminus just before the Xho I site.The fused genes were linked together by the second PCR using primersNgbEK and Cxholveg (for granzyme B-vegf121) or NgbEK and Czme2 (forgranzyme B-scFvMEL). In order to clone the fused genes into pET32a (+)vector with an enterokinase site at the N-terminus of granzyme B, thefragment from pET32a (+) was amplified by PCR using primers T7 promoter(5′ to 3′) TAATACGACTCACTATAG (SEQ ID NO:9) and CpET32EK (5′ to 3′)CTTGTCGTCGTCGTCGGTACCCAGATCTGG (SEQ ID NO:10). The primer has anenterokinase site at the C-terminus overlapping with the N-terminus offused gene. By overlap PCR, the fusion genes EK-Granzyme B-vegf121 wereconstructed using primers T7 promoter and CxhoIveg, and the fusion genesEK-Granzyme B-scFvMEL were constructed using primers T7 promoter andCzme2. The PCR reactions were performed by thirty cycles of 94° C. for 1min, 50° C. for 1 min and 72° C. for 1 min, with an extension reactionat 72° C. for 5 min. Amplified fragments were separated by 1% agarosegel electrophoresis and purified by PCR purification kit (Qiagen). Thepurified PCR products were digested with Xba I and Sho I at 37° C. for 3hrs and then separated by 1% agarose gel electrophoresis, purified fromthe gels and cloned into pET32a (+) vector, designated pET32GrB-vegf121or pET32GrB-scFvMEL. The ligation mixture was transformed into DH5αcompetent cells, the positive clones were screened by PCR, and thensequenced. A clone having the T7 promoter, lac operator, rbs, Trx.tag,His.tag, S-tag, and enterokinase sites to granzyme B-G4S-vegf121 orgranzyme B-G4S-scFvMEL, with no second site mutations, was chosen fortransformation into AD494 (DE3)pLysS competent cells for furtherinduction and expression.

Example 4 Methods—Induction and Expression of Granzyme B-Vegf121 orGranzyme B-scFvMEL Fusion Proteins in E. coli

The fusion constructs of Example 3 were induced and expressed asdescribed herein for utilization in multiple Examples elsewhere herein,including at least Examples 17 and 18. Bacterial colonies transformedwith the constructed plasmid were grown in Luria Broth (LB) growth mediacontaining 200 μg/ml ampicillin, 70 μg/ml chloramphenicol, and 15 μg/mlkanamycin, at 37° C. overnight at 240 rpm shaking. The cultures werethen diluted 1:100 in fresh LB media plus antibiotics and grown to A₆₀₀of 0.5 at 37° C. Thereafter, the cultures were induced by addition ofIPTG to a final concentration of 0.25 mM at 37° C. for 1.5 hrs. Thecells were harvested and resuspended in 10 mM Tris (pH 8.0) and storedfrozen at −80° C. for later purification.

Example 5 Methods—Purification of Granzyme B-Vegf121 or GranzymeB-scFvMEL Fusion Protein

The fusion constructs induced and expressed in Example 4 were purifiedas described herein for utilization in multiple Examples elsewhereherein, including at least Examples 17 and 18. The resuspension culturewas lysed by addition of lysozyme to a final concentration of 100 μg/mlwith agitation for 30 min at 4° C., which was followed by sonication.Extracts were centrifuged at 10,800 g for 30 min, and the supernatantwas further centrifuged at 40,000 rpm for 1 hr. The supernatantcontaining only soluble protein was adjusted to 40 mM Tris, pH 8.0, 10mM imidazole and applied to a nickel-NTA agarose equilibrated with thesame buffer. After washing the nickel-NTA agarose with 500 mM NaCl and20 mM imidazole, the bound proteins were eluted with 500 mM NaCl, and500 mM imidazole. Absorbance (280 nM) and SDS-PAGE analyses wereperformed to identify the polyhistidine-tagged protein, designatedPro-granzyme B-vegf121 or Pro-granzyme B-scFvMEL, respectively. Theeluted pro-granzymeB-vegf121 or pro-granzymeB-scFvMEL protein wasdialyzed against 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. Progranzyme Bmoiety of granzyme B-vegf121 or granzyme B-scFvMEL was activated by theaddition of recombinant bovine enterokinase (rEK) to remove thepolyhistidine-tag according to the manufacturer's instruction (1 unit ofrEK cleavage 50 μg protein, incubated at room temperature for 16 hrs).The rEK was removed by EK capture agarose. The final protein wasanalyzed by SDS-PAGE and stored at 4° C.

Example 6 Methods—SDS-PAGE and Western Blot Analysis

The following methods were performed for experiments as described in,for example, Example 15. Protein samples were analyzed byelectrophoresis on an 8.5% SDS-PAGE under reducing conditions. The gelswere stained with coomassie blue. For western blotting analysis,proteins were transferred from gels into nitrocellulose membranes. Themembranes were blocked with 5% non-fat milk and incubated for 1 hr atroom temperature with mouse anti-granzyme B monoclonal antibody (1.0μg/ml) or mouse anti-vegf121 polyclonal antibody (1:2000 dilution) orrabbit anti-scFvzme polyclonal antibody (1:2000 dilution). Afterwashing, the membranes were incubated with goat anti-mouse/horseradishperoxidase conjugate (HRP-GAM, 1:5000 dilution) or goatanti-rabbit/horseradish peroxidase conjugate (HRP-GAR, 1:5000 dilution).After further washing, the membrane was developed using the Amersham(Piscataway, N.J.) ECL detection system and exposed to X-ray film.

Example 7 Methods—Enzyme Assays

The enzymatic activity of granzyme B was determined in a continuouscolorimetric assay, with BAADT(N-α-t-butoxycarbonyl-L-alanyl-L-alanyl-L-aspartyl-thiobenzyl ester) assubstrate. Assays were performed in 200 μl and consisted of enzyme in100 mM HEPES, pH7.5, 10 mM CaCl₂, 1 mM 5,5′-dithiobis-2-nitrobenzoicacid, 0.2 mM substrate at 25° C. The change in absorbance at OD₄₀₅ wasmeasured on a Thermomax plate reader. Absorbance increases wereconverted to enzymatic rates by using an extinction coefficient of 13,100 cm-1M-1 that differed from the usual extinction coefficient of 13,600 cm-1M-1 at 412 nm reported by Ellman.

Example 8 Methods—Detection of scFvMEL Moiety of Granzyme B-scFvMEL

Reacti-Bind™ Protein L Coated Plates from PIERCE (Rockford, Ill.) wereused for detection of scFvMEL moiety of GranzymeB-scFvMEL, based onELISA method. Briefly, pre-coated Protein L was blocked by 5% BSA, andthe reaction was purified with Granzyme B-scFvMEL or other scFvMELfusion proteins at various concentrations, respectively. After washing,the proteins were incubated with rabbit ant-scFvZME antibody, followedby HRP-GAR, then substrate2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) solutionwith 1 μg/ml 30% H₂O₂ was added. Absorbance at 405 nm was measured after30 min.

Example 9 Methods—Cytotoxicity Assays In Vitro Against PAE-Flt-1 andPAE-Flk-1 for Granzyme B-Vegf121 and Against A375-M for GranzymeB-scFvMEL

PAE cells in Ham's F-12 medium with 10% FBS or A375-M cells in MEMmedium with 10% FBS were plated into 96-well plates at a density of2.5×10³ cells per well and allowed to adhere for 24 hr at 37° C. in 5%CO₂. After 24 hr, the medium was replaced with medium containingdifferent concentrations of granzymeB-vegf121 or granzyme B-scFvMEL.After 72 hr, the effect of granzymeB-vegf121 or granzymeB-scFvMEL on thegrowth of cells in culture was determined using crystal violet staining.Surviving adherent cells were stained by adding 100 μl of crystal violet(0.5% (w:v) in ethanol). The stain was incubated on the plates for 0.5hr, excess stain was removed, and the plates were washed with water andallowed to air-dry. The remaining dye was solubilized by addition of 150μl of Sornson's buffer (0.1 M sodium citrate, pH4.2). Plates were readon a microplate ELISA reader at 630 nM.

Example 10 In Vitro Transcription and Translation and In Vitro Cleavageof Procaspase 3 or DFF45 by Granzyme B or Bax Fusion Protein

An expression plasmid containing cDNA encoding procaspase-3 or DFF45will be linearized with a restriction endonuclease digested and³⁵S-labeled procaspase-3 or DFF45 protein will be generated using an invitro rabbit reticulocyte TNT kit (Promega) according to themanufacturer's instructions. In brief, the linear plasmid containingcDNA encoding procaspase-3 or DFF45 (1 μg) will be incubated with theTNT reaction mixture containing 25 μl of rabbit reticulocyte lysate, 2μl of TNT reaction buffer, 1 μl of T7 RNA polymerase, 1 mM amino acidmixture minus cysteine, 2 μl of [³⁵S] cysteine or 2 μl of [³⁵S]methionine (10 mCi/ml) and 40 U of RNase inhibitor (Amersham PharmaciaBiotech, Inc.) in a total volume of 50 μl for 90 min at 30° C. For invitro cleavage, the translation products will be incubated in thepresence of granzyme B fusion or Bax fusion proteins in 150 mM NaCl. Thereactions will be performed at 30° C. in a final volume of 10 μl forvarious time intervals. The reactions will be then stopped by theaddition of an equal volume of 2× Laemmli buffer. Cleavage products willbe then separated by 15% SDS-PAGE and detected either by immunoblottingor phosphorimaging of the dried gels.

Example 11 Apoptosis Assays

Cell Morphology:

A375-M or AAB527 cells (for Granzyme B-scFvMEL) and PAE-Flk1 or PAE-Flt1cells (for Granzyme B-vegf121) will be grown in appropriate cell culturemedia. Cell death will be monitored by XTT assay. To visually monitorgranzyme B-mediated or Bax-mediated apoptosis of these cells, 1×10⁴cells will be plated in each well of a 12-well microscope slide.Forty-eight hours later, cells will be washed once in PBS, then treatedwith 25 μl of serum-free medium supplemented with 1 μg/ml DTT andgranzyme B fusion proteins or Bax fusion proteins. Following incubationat 37° C. for 1 hr, supernatants will be removed and replaced with 50 μlof complete medium. After a further 2 h at 37° C., cells will be gentlywashed with PBS and fixed with acetone: methanol (1:1) for 2 min at roomtemperature. Apoptosis of adherent cells will be visualized byphase-contrast microscopy.

Assay of DNA Fragmentation:

To monitor DNA fragmentation, 5×10⁵ cells in 50 μl of serum-free mediumwill be supplemented with 1 μg/ml DTT and granzyme B fusion proteins orBax fusion proteins. Following incubation at 37° C. for 1 hr, they willbe washed once in PBS. Fragmented DNA will be extracted using aphenol/chloroform extraction assay. Briefly, the cell pellet will bere-suspended in 25 μl of PBS, and an equal volume ofpheno/chloroform/isoamylalcohol (1:1:0.1) added. Following gentleagitation and centrifugation (10,000 g for 2 min), fragmented DNA willbe recovered, treated with RNase A for 1 hr at 37° C. and analyzed on 2%agarose gels containing ethidium bromide.

FACS Analysis:

Cells (5×10⁵/10 ml) will be centrifuged at 450×g for 6 min, washed withcold PBS and resuspended in 300 μl of PBS. The cells will be fixed with5 ml methanol and left at −20° C. for at least 1 hr. The cells will bethen centrifuged at 800×g for 5 min., resuspended in 100 μl PBS anddiluted to a final volume of 1 ml with PBS. Cells will be incubated onice for an additional 30 min, centrifuged at 800×g for 5 min andresuspended in 0.5 ml PBS. 10 μl RNase (50 μg/ml) and propidium iodide(PI, 5 μg/ml) will be added to the cell samples which will be then FACSanalyzed for DNA content as a function of cell number.

Cleavage of Caspase-3, PARP and DFF Detected in Cell Samples Treatedwith Fusion Proteins vs. Non-Treated with Fusion Proteins withinDifferent Time Course or Dose by Using Western Blot Analysis:

For western blotting, samples will be separated by electrophoresis using14% SDS-PAGE. The proteins will be transferred from gels intonitrocellulose membranes. The membranes will be blocked with 5% non-fatmilk, and incubated for 1 hr at room temperature with anti-caspase-3 orcleaved caspase-3, or anti-PARP or anti-cleaved DFF antibody (obtainedfrom Cell Signaling Technology). After washing, the membranes will beincubated with goat anti-rabbit or goat anti-mouse/horseradishperoxidase conjugate. After further washing, the membranes will bedeveloped using the Amersham ECL detection system and exposed to X-rayfilm.

Example 12 Signal Transduction Pathway-Non-Apoptosis Assays

In the following example, experiments are described which are performedto analyze insulin signal transduction pathways, which are non-apoptoticpathways, in cells treated with a delivery vehicle containing proteinkinase B, which is a critical point in the insulin signal transductioncascade leading to modulation of the enzyme glycogen synthetase kinase-3(GSK-3). In the present invention, a fusion construct of the cytokinehuman hepatocyte growth factor (HCF) and the signal transductionregulator protein kinase B(PKB) will be generated. The HCF componentserves to bind to hepatocytes and to deliver active PKB to the cellularcytoplasm. From there, activation of the downstream modulators ofinsulin signaling (GSK-3) will be assessed as described below.

Anti-GSK3 antibodies are obtained from Transduction Laboratory.Phosphotyrosine antibody 4G10 and anti-PKB antibody will be obtainedfrom Upstate Biotechnology. Phosphospecific antibody against Ser-9 ofGSK3 will be obtained from Quality Controlled Biochemicals.

Cultures of hepatocytes in media will be treated with various doses ofHCF or the HCF/PKB fusion construct. At various times after drugaddition, cells will be harvested. To prepare cytosolic fractions, cellswill be washed and collected in ice-cold phosphate-buffered saline. Cellpellets will be resuspended in ice-cold hypotonic buffer (25 mM Tris, pH7.5, 1 mM EDTA, 25 mM NaF, 1 mM dithiothreitol) with Complete proteaseinhibitor mixture (Roche Molecular Biochemicals; Indianapolis, Ind.).Cells will be lysed after incubating on ice for 10 min (verified bymicroscope analysis). The lysates will be subjected toultracentrifugation at 100,000×g for 30 minutes at 4° C., and thesupernatant will be collected. For immunoprecipitation, cells will bewashed twice in ice-cold phosphate-buffered saline, and then lysed in IPbuffer (125 mM NaCl, 25 mM NaF, 25 mM Tris, pH 7.5, 1 mM EDTA, 1 mMEGTA, 1% Triton X-100, 10 mM-glycerol phosphate, 5 mM sodiumpyrophosphate, 1 mM NaVO3, 200 nM okadaic acid, 1 mM dithiothreitol)with Complete protease inhibitor mixture. Anti-GSK3 antibody will beadded to clarified lysates for 1 h at 4° C., and then Protein G beads(Sigma) will be added for another 1 h. Immunoprecipitates will be washedthree times with IP buffer.

For GSK3 kinase assays, cells treated with the delivery vehiclecontaining active protein kinase B fusion construct GSK3immunoprecipitates will be washed once with kinase buffer (25 mM Tris,pH 7.5, 10 mM MgCl2) first. Kinase reactions will be performed in kinasebuffer with 100 μM [γ-³²P]ATP and 100 μM 2BSP peptide as the substrate(synthesized by the Biomedical Resource Center, University ofCalifornia, San Francisco, Calif.). 2BSP is based on the GSK3 targetsite in eIF2B. After 20 min at 30° C., the reactions will be spotted onphosphocellulose P81 paper (Whatman), washed four times with 100 mMphosphoric acid, and counted in scintillation counter.

Example 13 Human Granzyme B Gene Cloning from Hut78

Native human granzyme B is a cytotoxic lymphocyte granule serineproteinase produced by cytotoxic T cells and natural killer cells.Initial attempts to clone human granzyme B gene from HL-60 cells, whichare promyelocytic leukemia cells from human peripheral blood, wereunsuccessful. However, the targeted cDNA, as a pre-mature granzyme Bgene, was obtained from human cutaneous T cell lymphoma Hut78 cells, byisolating RNA and using RT-PCR. A 1% agarose gel electrophoresis showedthat human pre-mature granzyme B cDNA was ˜800 bp (FIG. 1). The genesequence and amino acid sequence (FIG. 2) showed that the first 20 aminoacids are signal sequence. The human granzyme B sequence with signalsequence is designated pre-mature granzyme B. In cytotoxic cells, activegranzyme B is generated from a zymogen by dipeptidyl peptidase I(DPPI)-mediated proteolysis (Smyth et al., 1995). This removes thetwo-residue (Glyl⁹Glu²⁰) propeptide and exposes Ile²¹ to become themature, N-terminal Ile-Ile-Gly-Gly sequence granzyme B.

Example 14 Construction of Granzyme B-Vegf121 or Granzyme B-scFvMEL

PCR was used to amplify the coding sequence of granzyme B from Ile²¹,which is the first residue of the mature enzyme, effectively deletingthe signal sequence and GlyGlu domain. Concomitantly, a cleavage sitewas inserted for enterokinase (AspAspAspLys; SEQ ID NO:53) upstream andadjacent to Ile²¹. Granzyme B was attached to the recombinant Vegf121 orscFvMEL via flexible tether (G4S). The fused gene fragment was thenintroduced into the Xba I and Xho I sites of the pET32a (+) to form theexpression vector pET32GrB-vegf121 (FIG. 3A) and pET32GrB-scFvMEL (FIG.3B). This vector contains a T7 promoter for high-level expressionfollowed by a Trx.tag, a His.tag, a thrombin cleavage site, and anenterokinase cleavage site for final removal of the protein purificationtag (FIGS. 4A and 4B). Once the protein tag is removed by recombinantenterokinase, the first residue Ile of mature granzyme was exposed, andthe granzyme B moiety of granzyme B-vegf121 or granzyme B-scFvMEL wasactivated. The nucleotide sequences and amino acid sequences of granzymeB-vegf121 (1059 base pairs, 353 aa) (FIGS. 4C and 4D) and granzymeB-scFvMEL (1440 base pairs, 480 aa) (FIGS. 4E and 4F) were confirmed.

Example 15 Expression and Purification of Granzyme B-Vegf121 or GranzymeB-scFvMEL Fusion Protein

The recombinant protein granzyme B-vegf121 or granzyme B-scFvMEL wasexpressed as polyhistidine-tagged protein designed pro-granzymeB-vegf121or pro-granzyme B-scFvMEL and then purified by Nickel-NTA metal affinitychromatography. The his-tag was cleaved by addition of rEK to formgranzymeB-vegf121 or granzyme B-scFvMEL. One liter of the culturetypically yielded approximately 100 μg of the final purifiedgranzymeB-vegf121 product and 150 μg of the final purified granzymeB-scFvMEL product.

The results showed induced expression of granzyme B-related fusionconstructs. The induced band at ˜55 kDa for granzyme B-vegf121 and at˜72 kDa for Granzyme B-scFvMEL represent, respectively, the granzymeB-vegf121 or granzyme B-scFvMEL construct containing a ˜18 kDapurification tag. Enzymatic digestion of the tag using recombinantenterokinase (rEK) resulted in appearance of a band migrating at ˜38 kDafor granzyme B-vegf121 and at ˜53 kDa for granzyme B-scFvMELrepresenting native proteins. Thus, SDS-PAGE showed that the finalpurified granzymeB-vegf121 fusion construct migrated under reducingconditions as a band at the expected molecular weight of 38 kDa (FIG.5A) and granzyme B-scFvMEL fusion construct showed the band at theexpected molecular weight of 53 kDa (FIG. 5B).

Specificity of the cleaved fusion protein was confirmed by Western blotusing either mouse anti-granzyme B monoclonal antibody, mouseanti-vegf121 polyclonal antibody, or rabbit anti-scFvZME polyclonalantibody (FIG. 6). The results showed that granzyme B-vegf121 fusionconstruct (FIG. 6A) could specifically bind to either mouse anti-vegf121antibody (FIG. 6A (a)) or mouse anti-granzyme B monoclonal antibody(FIG. 6A (b)). The molecular weights of pro-granzyme B-vegf121 andGranzyme B-vegf121 are approximately 55 kDa and 38 kDa, respectively.Granzyme B-scFvMEL fusion construct (FIG. 6B) could specifically bind toeither mouse anti-granzyme B monoclonal antibody (FIG. 6B (c)) or rabbitanti-scFvMEL polyclonal antibody (FIG. 6B (d)). The molecular weights ofPro-granzyme B-scFvMEL and Granzyme B-scFvMEL are approximately 70 kDaand 53 kDa, respectively.

Example 16 Binding Activity of scFvMEL Moiety of Granzyme B-scFvMELFusion Protein

The scFvMEL moiety was tested to bind Protein L, which is animmunoglobulin-binding protein that originally comes from the bacteriaPeptostreptococcus magnus. Protein L has the unique ability to bindthrough kappa light chain interactions without interfering with anantibody's antigen-binding site. This gives Protein L the unique abilityto bind Single Chain Variable Fragments (scFv). The results showed theabsorbance at 405 nm concentration-response increase, suggesting scFvMELbound to Protein L. (FIG. 7). The binding activity of the granzymeB-scFvMEL was the same as that of the scFvMEL-TNF, which couldspecifically bind to antigen-positive human melanoma cells and wascytotoxic activity to those melanoma cells.

Example 17 In Vitro Cytotoxic Effects of Granzyme B-Vegf121

The cytotoxicity of GranzymeB-vegf121 was assessed against log-phasePAE-Flk-1 (Overexpression flk-1/KDR receptor) and PAE-Flt-1(overexpression flt-1 receptor) in culture, wherein 2.5×10³ cells perwell on 96-well plates. A 50% growth inhibitory effect was found at aconcentration of ˜10 nM on PAE-Flk-1 cells. However, no cytotoxiceffects were found on PAE-Flt-1 cells (FIG. 8). It was also shown thatVEGF121 could specifically bind to VEGF receptor Flk-1/KDR but not toFlt-1. The cytotoxicity of granzymeB-vegf121 demonstrated that theconstruct could specifically kill PAE-Flk-1 cells, which indicated thatthe Vegf121 moiety of the fusion bound to the Flk-1 over-expressioncell-surface. Subsequently, there was delivery of granzyme B to theinterior of targeted cells, resulting in cytotoxicity to the targetcells.

Example 18 In Vitro Cytotoxic Effects of Granzyme B-scFvMEL

The cytotoxicity of granzyme B-scFvMEL was tested against log-phasehuman melanoma A375-M cells. The results showed that granzyme B-scFvMELcould kill the A375-M cells, with an IC₅₀ concentration of approximately20 nM. When pre-treated with scFvMEL-3825 at the concentration of 178.5nM for 6 hr, followed by treatment with granzyme B-scFvMEL for 72 hr, a15-fold higher concentration of granzyme B-scFvMEL was required toexhibit 50% cytotoxicity compared to the absence of scFvMEL-3825pre-treatment (FIG. 9). In a specific embodiment, this is because thecell-surface antigen gp240 was already occupied by scFvMEL-3825,resulting in a reduced chance for the scFvMEL moiety of granzymeB-scFvMEL binding to the gp240 antigen, consequently inhibiting thecytotoxicity of granzyme B-scFvMEL on these cells. The results suggestedthat the cytotoxicity, at least in part, is due to the interaction ofthe antibody with its cell-surface domain.

Example 19 Cloning Human Bax Gene

Total RNA from Namalwa cells was isolated using Glass MAX RNAMicroisolation Spin Cartridge System (Gibco BRL). Removal of the genomicDNA was performed by addition of RNase-free DNase I while incubating atroom temperature for 15 min. DNase I then was inactivated by adding EDTAsolution and heating for 15 min at 65° C. SUPERSCRIPT First-StrandSynthesis System was utilized for RT-PCR (Gibco BRL). First-strandsynthesis used Oligo (dT), and then the target cDNA (Bax cDNA) wasamplified using the primers: NbaxTA (5′ to 3′): GGTGATGGACGGGTCCGGGGAGCA(SEQ ID NO:29) and CbaxTA (5′ to 3′): GGCCTCAGCCCATCTTCTTCCAGATGGTGA(SEQ ID NO:30) by PCR with the following cycles: denaturation at 95° C.for 5 min, 30 cycles of 94° C. for 1 min, 50° C. for 1 min, 72° C. for 1min, and extension at 72° C. for 5 min. A 1% agarose gel was run tocheck the PCR product. Purified PCR fragment was cloned into PCR 2.1 TAvector (Invitrogen) and designed BaxTA. The BaxTA was transformed intoINVαF′ competent cells, and the positive clones were screened byblue/white colonies screening or by PCR methods. The DNAs for positiveclones were isolated by using QIApre Spin prep kit (Qiagen; Valencia,Calif.), and sequencing confirmed the human bax gene in the correctclone (BaxTA-35 (clone # 35)).

Example 20 Construction of Bax-Related Fusion Genes

The construction was based on an over-lap PCR method. The scFvMEL geneswere fused to Bax, truncated Bax1-5 (that is, comprises exons 1 through5) or truncated Bax345 (that is, comprises exons 3, 4, and 5) genes withG4S tether in different orientation (designated scFvMEL-bax orBax-scFvMEL, scFvMEL-Bax1-5 or Bax1-5-scFvMEL, and scFvMEL-Bax345 orBax345-scFvMEL, respectively). As shown in FIG. 10, a skilled artisanrecognizes that the human Bax gene (SEQ ID NO:45), which encodes thepolypeptide of SEQ ID NO:46, comprises six exons, with the domain BH1(DGNFNWGRVVA; SEQ ID NO:47) in exon 4, BH2 (WIQDQGGWD; SEQ ID NO:48) inexon 5, and BH3 (LKRIGDE; SEQ ID NO:49) in exon 3. In a specificembodiment, the Bax chimeric polypeptide comprises the BH1, BH2 and BH3domains or a combination thereof. In another specific embodiment, theBax chimeric polypeptide consists essentially of the BH1, BH2 and BH3domains. In another specific embodiment, the Bax chimeric polypeptidecomprises exons 3, 4, and 5 or a combination thereof. In an additionalspecific embodiment, the Bax chimeric polypeptide consists essentiallyof exons 3, 4, and 5.

Briefly, scFvMEL coding sequence was amplified from pET32a-scFvMEL/TNFby PCR and full length bax or truncated Bax1-5 or truncated Bax345 wasamplified from BaxTA-35 by PCR. Different primers were designed whereinG4S liner sequence was added to the C-terminus or N-terminus in order tolink the fused genes together by the second PCR. In order to clone thefused genes into pET32a (+) vector at Nco I and Xho I sites, primersadded the Nco I site at the N-terminus and two stop codons, and a Xho Isite was added at the C-terminus. The first PCR were performed by 95° C.for 5 min, 30 cycles of 94° C. for 1 min, 50° C. for 1 min and 72° C.for 1 min, and then extension at 72° C. for 5 min. For constructing ofscFvMEL-bax, scFvMEL was amplified by using primers: NcoIzme (5′ to 3′):GGTGGCGGTGGCTCCATGGCGGACATTGTGATGACCCAGTCTCAAAAATTC (SEQ ID NO:31) andCzme (5′ to 3′): CGTCGGAGCCACCGCCACCGCTAGCTGAGGAGACGGTGAGAGT (SEQ IDNO:32), Bax fragment was amplified by using primers: Nbax2 (5′ to 3′):GGTGGCGGTGGCTCCGACGGGTCCGGGGAGCAG (SEQ ID NO:33) and Cbax (5′ to 3′):GGAGCCACCGCCACCCTCGAGCTATCAGCCCATCTTCTTCCAGAT (SEQ ID NO:34). ForBax-scFvMEL construct, primers are Nbax (5′ to 3′):GGTGGCGGTGGCTCCATGGACGGGTCCGGGGAGCAG (SEQ ID NO:35), Cbax2-1 (5′ to 3′):GTCCGTGGAGCCACCGCCACCGCTAGCGCCCATCTTCTTCCA (SEQ ID NO:36), Nzme2 (5′ to3′): GGTGGCGGTGGCTCCACGGACATTGTGATGACCCAGTCTCAAAAATTC (SEQ ID NO:37) andCzme2 (5′ to 3′): GGAGCCACCGCCACCCTCGAGCTATCATGAGGAGACGGTGAGAGTGGT (SEQID NO:38). For construction of scFvMEL-Bax1-5 construct, primers areNcoIzme, Czme, Nbax2 and CxhoIbax345 (5′ to 3′):GGAGCCACCGCCACCCTCGAGCTATCACCAACCACCCTGGTC (SEQ ID NO:39). Forconstruction of Bax 1-5-scFvMEL fusion construct, primers are Nbax,Cbax345 (5′ to 3′): GGAGCCACCGCCACCCCAACCACCCTGGTC (SEQ ID NO:40), Nzme2and Czme2. For construction of scFvMEL-Bax345fusion, primers areNcoIzme, Primer3 (5′ to 3′): CCGGAGCCACCGCCACCGCTAGCTGAGGAGACTGTGA (SEQID NO:41), Nbax345 (5′ to 3′): GGTGGCGGTGGCTCCTTCATCCAGGATCGAG (SEQ IDNO:42), and CxhoIbax345. For construction of Bax345-scFvMEL, NcoIBax345(5′ to 3′) is used: GGTGGCGGTGGCTCCATGGTCATCCAGGATCGAG (SEQ ID NO:43),Cbax345, Nzme2 and Czme2.

Then, the second PCR was performed by 30 cycles of 94° C. for 1 min, 50°C. for 1 min and 72° C. for 1 min, further extension at 72° C. for 5min. Amplified fragments were separated by 1% agarose gelelectrophoresis, purified by PCR purification kit (Qiagen; Valencia,Calif.). The purified PCR products were digested with Nco I and Xho I at37° C. for 3 hrs and then separated by 1% agarose gel electrophoresis,purified from the gels using geneclean II kit (Quantium Biotechnologies,Inc., Carlsbad, Calif.). The purified gene fragments were cloned intopET32a (+) vector, the ligation mixture was transformed into DH5αcompetent cells, screened the positive clones by PCR, then sequenced.The clones with correct-frame sequence were transformed into AD494(DE3)pLysS competent cells for further induction and expression.

For expression of full length Bax and Bax-scFvMEL protein, the Bax andBax-scFvMEL sequences were subcloned into pBAD/His A vector anddesignated pBAD/Hisbax and pBAD/Hisbax-scFvMEL, respectively. Briefly,the full length Bax and Bax-scFvMEL genes were amplified by using a PCRmethod. For amplification of full length Bax, BaxTA-35 was used astemplate, and the primers were NBAXHIS (5′ to 3′):AAACATGCCATGGCTCACCACCACCACCACCACGACGGGTCCGGGGAGCAGCCC AGA (SEQ IDNO:44) and Cbax. For amplification of Bax-scFvMEL, pET32-Bax-scFvMEL(clone2) was used as template, and primers were NBAXHIS and Czme2. TheNBAXHIS primer was designed as follows: Nco I site for cloning,polyhistidine (6×His) for purification and detection at the N-terminus,followed by the initiation ATG; two stop codons and a Xho I site wereadded at the C-terminus in Cbax and Czme2 primers. Purified PCRfragments were digested by Nco I and Xho I and purified by using geneclean kit, following ligation into the same restriction endonucleasesdigested for the pBAD/His A vector. The ligation mixture was transformedinto DH5α, and the positive clones were screened by PCR screening, DNAwas isolated, and the sequence was checked. The confirmed sequencepositive clones were transformed into LMG194 competent cells forexpression.

Example 21 Induction and Expression of Bax-Related Fusion Proteins in E.Coli

Bacterial colonies transformed with the constructed plasmid were grownin Luria Broth (LB) growth media containing 200 μg/ml ampicillin, 70μg/ml chloramphenicol, and 15 μg/ml kanamycin, at 37° C. overnight at 24rpm shaking. The cultures were then diluted 1:100 in fresh LB media plusantibiotics and grown to A₆₀₀=0.6 at 37° C., thereafter, induced byaddition of IPTG to a final concentration of 80 μM at 37° C. for 2 hrs.The cells were harvested, resuspended in 10 mM Tris (pH 8.0) and storedfrozen at −80° C. for further purification.

Example 22 Induction and Expression of Full Length Bax and Bax-scFvMEL

Bacterial colonies transformed with the plasmid pBAD/Hisbax andpBAD/Hisbax-scFvMEL were grown in RM medium containing glucose and 100μg/ml ampicillin, at 37° C. overnight with shaking (225 rpm). Thecultures were then diluted by 1: 100 in fresh RM medium containingglucose and 100 μg/ml ampicillin and grown at 37° C. with shaking tomaxium OD₆₀₀, then induced by addition of 5% arabinose at 37° C. for 4hours. The cells were harvested, resuspended in 10 mM Tris (pH 8.0) andstored frozen at −80° C. for further purification.

Example 23 Purification of Bax-Related Fusion Proteins

Re-suspension was lysed by addition of lysozyme to a final concentrationof 100 μg/ml tumbling for 30 min at 4° C., followed by sonication.Extracts were centrifuged at 10,800 g for 30 min, and the supernatantwas further centrifuged at 40,000 rpm for 1 hr. The supernatantcontaining only soluble protein was adjusted to 40 mM Tris, pH 8.0, 10mM Imidazole and was applied to a Nickel-NTA agarose equilibrated withthe same buffer. After washing the Nickel-NTA agarose with 500 mM NaCl20 mM imidazole, the bound proteins were eluted with 500 mM NaCl 500 mMimidazole. Absorbance (280 nm) and SDS-PAGE analysis were performed todetermine the polyhistidine-tagged proteins. The final proteins wereobtained by the addition of recombinant bovine enterokinase (rEK) toremove the polyhistidine-tag according to the manufacturer's instruction(1 unit of rEK cleavage 50 μg protein, incubated at room temperature for16 hrs). The rEK was then removed by EK capture agarose. The finalproteins were analyzed by SDS-PAGE and stored at 4° C.

Example 24 SDS-PAGE and Western Blot Analysis

Protein samples were analyzed by electrophoresis on a 8.5% or 12%SDS-PAGE under reducing conditions. The gels were stained with CoomassieBlue. For western blotting analysis, proteins were transferred from gelsinto nitrocellulose membranes. The membranes were blocked with 5%non-fat milk and incubated for 1 hr at room temperature or overnight at4° C. with rabbit anti-scFvzme antibody (1:2000 dilution) or rabbitanti-bax antibody (1:1000 dilution). After washing, the membranes wereincubated with goat anti-rabbit/horseadish peroxidase conjugate(HRP-GAR, 1:5000 dilution). After further washing, the membrane wasdeveloped using the Amersham ECL detection system and exposed to X-rayfilm.

Example 25 Detection of scFvMEL Moiety of Bax-Related Fusion Proteins

Reacti-Bind™ Protein L Coated Plates from PIERCE (Rockford, Ill.) or96-well plates containing adherent human melanoma A375-M cells were usedfor detection of scFvMEL moiety of Bax-related fusion proteins, based onELISA method. Briefly, Pre-coated Protein L was blocked by 5% BSA andthen reacted with scFvMEL bax-related fusion proteins at variousconcentration. After washing, they were incubated with rabbitanti-scFvZME antibody, followed by incubation with HRP-GAR (1:5000dilution) for 1 hr at room temperature, and then substrate 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) solution with1 μg/ml 30% H₂O₂ added. Absorbance at 405 nm was measured after 30 min.

Example 26 In Vitro Cytotoxicity Assays

Human melanoma A375-M cells cultured in MEM medium with 10% FBS wereplated into 96-well plates at a density of 2.5×10³ cells per well andallowed to adhere for 24 hr at 37° C. in 5% CO₂. After 24 hr, the mediumwas replaced with medium containing different concentrations ofdifferent scFvMEL-bax-related fusion proteins. After 72 hr, the effectof those fusion proteins on the growth of cells in culture wasdetermined using crystal violet staining, or XTT. Plates were read on amicroplate ELISA reader at 540 nm.

Example 27 Cytotoxicity of Bax Chimeric Polypeptides

As demonstrated in FIG. 11, the human bax gene was cloned by PCR fromNamalwa cells. A 1% agarose gel electrophoresis demonstrating human BaxcDNA synthesized from Namalwa cells by RT-PCR.

FIGS. 12A and 12B illustrate construction of scFvMEL Bax-related fusionproteins. The Bax gene consists of six exons, and the gene producesalternative transcripts, the predominant form of which encodes a 1.0 kbmRNA and transcript 21 kDa protein which designated Bax α. The boxesindicate exons identified by numbers. Exon 6 is the transmembranedomain. The scFvMEL genes were fused to Bax, truncated Bax 1-5 or Bax345with G4S tether in two different orientations, designated scFvMEL-bax,Bax-scFvMEL, scFvMEL-bax1-5, Bax1-5-scFvMEL, scFvMEL-bax345 andBax345-scFvMEL. The fusion genes were cloned into pET32a(+) expressionvector at Nco I and Xho I sites. Then the plasmid containing fusiongenes was transformed into AD494(DE3)pLysS E. coli for expression.

FIG. 13 demonstrates western blot analysis illustrating expression ofscFvMEL Bax-related fusion proteins in pET32a (+) vector. The SDS-PAGECoomassie Blue staining of truncated bax-related proteins occurred underreducing conditions. The results showed induced expression of scFvMELand truncated bax fusion proteins in pET32a(+) expression vector. Theinduced bands were at ˜62 kDa for scFvMEL-bax345 and Bax345-scFvMEL andwere at ˜65 kDa for scFvMEL-Bax1-5 and Bax1-5-scFvMEL, respectively,which also contained a ˜20 kDa purification tag.

FIG. 14 shows the expression of pET32-scFvMEL-bax and pET32-Bax-scFvMELtransformed into AD494(DE3)pLysS E coli and under IPTG induction. Thefull-length bax fusion proteins are very toxic to the bacteria becauseof the highly hydrophobic domain-exon 6. The bacteria containing theplasmid pET32-scFvMEL-Bax or pET32-Bax-scFv were grown in LB mediacontaining 200 μg/ml ampicillin, 70 μg/ml chloramphenicol and 15 μg/mlkanamycin to OD₆₀₀=0.6, induced by addition of IPTG to the finalconcentration of 0.1 mM, the bacteria died in terms of the decreasevalue of OD₆₀₀.

FIG. 15 demonstrates the expression of the full length bax andBax-scFvMEL in pBAD/HisA vector transformed LMG194 E coli. The fulllength bax gene and Bax-scFvMEL gene were cloned into pBAD/His A vectorat Nco I and Xho I sites. The polyhistidine (6 his) tags were followedby the initiation ATG. The plasmids containing either Bax or Bax-scFvMELwere transformed into LMG194 cells for expression, and the expression ofthe Bax and Bax-scFvMEL proteins was tested in different bacterialgrowth media (RM containing glucose and 100 μg/ml ampicillin, RMcontaining 100 μg/ml ampicillin, and LB containing 100 μg/mlampicillin). The LMG194 transformed pBAD/HisLacZ was used as a positivecontrol, with an expression band at 120 kDa representing LacZ proteinwhich could be detected by anti-bax antibody. The LMG194 transformedpBAD/HisA (empty vector) was used as a negative control. Westernblotting was performed using rabbit anti-Bax monoclonal antibody (1:1000dilution) as the primary antibody and HRP-Goat-anti-rabbit IgG as thesecondary antibody. The results showed that the bands at ˜21 kDarepresent Bax with 6×His-tag, and the bands at 49 kDa representBax-scFvMEL with 6×His-tag.

Binding activity of scFvMEL moiety of the fusion proteins isdemonstrated in FIGS. 16A and 16B. A375-M cells are gp240antigen-positive human melanoma cell lines. Protein L has the uniqueability to bind scFv. The fusion protein Bax345-scFvMEL could bind toeither A375-M (FIG. 16A) or Protein L (FIG. 16B) detected withanti-scFvzme antibody. This binding activity is the same as the otherfusion protein scFvMEL-TNF.

FIG. 17 demonstrates cytotoxicity of scFvMEL-bax345 vs. Bax345-scFvMELon A375-M. The cytotoxic effects of scFvMEL-bax345 and Bax345-scFvMELagainst log-phase human antigen-positive melanoma A375-M cells wereassessed. A375-M cells were set up in 96-well plates (2.5×10³ cells perwell). The IC₅₀ concentrations of scFvMEL-bax345 and Bax345-scFvMEL wereapproximately 3 nM and 10 nM, respectively.

Example 28 Elisa of Granzyme B-Vegf121 on Various Cell Lines (Detectedwith Mouse Anti-Vegf121 Antibody)

ELISA Assay of Binding Activity utilized 96-well plates containingadherent PAE/flk-1, PAE/flt-1, A375-M or SKBR3-HP cells that wereblocked by 5% BSA and then reacted with purified Granzyme B-vegf121 atvarious concentrations. After washing, they were incubated with mouseanti-vegf antibody, followed by HRP-labeled goat anti-mouse IgG. Then asa substrate 2, 2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)solution with 1 μl/ml of 30% H₂O₂ was added. Absorbance at 405 nm wasmeasured after 30 min.

VEGF121 can specifically bind to the flk-1/KDR receptor on the vascularendothelial cells (FIG. 18). The experiment showed that the fusionprotein Granzyme B-vegf121 specifically binds to PAE/flk-1 cells whichoverexpress flk-1 receptors, but there were no binding activities onPAE/flt-1 (overexpressed flt-1 receptors) or human melanoma A375-M cellsand human breast cancer SKBR3-HP cells. That is, GrB/VEGF121 canspecifically bind to PAE/flk-1 cells which overexpress flk-1/KDRreceptor detected with either VEGF121 antibody or GrB antibody

Example 29 Cytotoxicity of Granzyme B/VEGF121 on Transfected EndothelialCells

Cytotoxicity of Granzyme B-VEGF121 against PAE/flk-1 vs PAE/flt-1 cellswas assessed against log-phase PAE/flk-1 (transfected with flk-1/KDRreceptor) and PAE/flt-1 (transfected with flt-1 receptor) in culture.Briefly, PAE cells were plated into 96-well plates at a density of2.5×10³ cells per well. After 24 hr, the cells were treated with mediumcontaining different concentrations of Granzyme B-VEGF121. The effect onthe growth of cells was determined using XTT after 72 hrs. Plates wereread on a microplate ELISA reader at 540 mm.

The results showed that a 50% growth inhibitory effect (I.C.₅₀) wasfound at a concentration of ˜10 nM on PAE/flk-1 cells (FIG. 19).However, no cytotoxic effects were found on PAE/flt-1 cells. Thecytotoxicity of Granzyme B-VEGF121 on PAE/flk-1 cells indicates that theVEGF121 moiety of the fusion specifically bound to flk-1 over-expressionon the cell-surface, followed by delivery of granzyme B to the interiorof targeted cells and cytotoxicity to the target cells.

Example 30 Cytotoxicity Assay of Granzyme B-Vegf121 Vs Vegf121-Rgel inVitro Against PAE/Flk-1

PAE cells in Ham's F-12 medium with 10% FBS were plated into 96-wellplates at a density of 2.5×10³ cells per well and allowed to adhere for24 hr at 37° C. in 5% CO₂. After 24 hr, the medium was replaced withmedium containing different concentrations of Granzyme B-VEGF121 orVEGF121-rGel. After 72 hrs, the effect of Granzyme B-VEGF121 orVEGF121-rGel on the growth of cells in culture was determined using XTT.Plates were read on a microplate ELISA reader at 540 nm.

The results showed that the I.C.₅₀ of VEGF121-rGel was approximately 1nM (FIG. 20). The I.C.₅₀ of Granzyme B-VEGF121 was 10-fold higher thanthat of VEGF121-rGel.

Example 31 Caspase Activity on PAE Cells Treated with Granzyme B-Vegf121

Western Blotting analysis of caspase activation was carried out with 30μg of whole cell lysates. Following SDS-PAGE, the proteins wereelectrophoretically transferred onto nitrocellulose membranes. Themembranes were blocked with phosphate-buffered saline with 0.5% Tween 20(PBST) containing 5% fat-free milk and then exposed to caspase-8,caspase-3, caspase-6, caspase-7, cleaved caspase-3, PARP or cleavedDFF45 antibodies, respectively. The membranes were washed with PBST andtreated with secondary antibodies conjugated to horseradish peroxidase.The antigen-antibody reaction was visualized by an enhancedchemiluminescence (ECL) assay using Amersham ECL develop reagents andexposure to film.

The results showed that after a 4 hr treatment by Granzyme B-VEGF121,cleaved caspase-8, cleaved caspase-3, cleaved PARP and cleaved DFF45were observed on PAE/flk-1 cells but not on PAE-flt-1 cells (FIG. 21).However, caspase-6 or caspase-7 was not cleaved by Granzyme B-VEGF121,which indicated that Granzyme B-VEGF121 activated caspases only involvedin caspase-8, caspase-3 apoptosis pathway.

Example 32 In Situ Cell Death Detection (Tunel)

Cleavage of genomic DNA during apoptosis may yield double-stranded, lowmolecular weight DNA fragments as well as single strand breaks (nicks)in high molecular weight DNA. Those DNA strand breaks can be identifiedby labeling free 3′-OH termini with modified nucleotides in an enzymaticreaction. This method uses terminal deoxynucleotidyl transferase (TdT),which catalyzes polymerization of nucleotides to free 3′-OH DNA ends ina template-independent manner, is used to label DNA strand breaks.Incorporated fluorescein is detected by anti-fluofescein antibody Fabfragments from sheep, conjugated with alkaline phosphatase (AP). Aftersubstrate reaction, stained cells can be analyzed under lightmicroscope.

For the cell treatments, cells were plated onto 16-well chamber slides,2500 cells/well, incubated for overnight at 37° C./5% CO₂ conditions.Cells were treated with fusion protein GrB-scFvMEL or GrB-vegf121 atI.C.₅₀ concentration for different times (24, 48 hr, etc.) and washedbriefly with PBS.

For the TUNEL Assay, cells were fixed with 3.7% formaldehyde at roomtemperature for 20 min, after rinsing with PBS, permeabilzed with 0.1%Triton X-100, 0.1% sodium citrate on ice for 2 min and then washed withPBS twice. Cells were incubated with TUNEL reaction mixture at 37° C.for 60 min, followed by incubation with Converter-AP at 37° C. for 30min, and finally reacted with Fast Red substrate solution at roomtemperature for 10 min. After final wash step, the slides were mountedin mounting medium and analyzed under light microscope.

Positive controls were included in each experimental set up. Fixed andpermeabilized cells were incubated with 1 mg/ml of DNase I for 10 min at37° C. to induce DNA strand breaks.

The effect of GrB-scFvMEL on A375-M and SKBR3-HP cells by in situ celldeath detection (TUNEL) was determined. TUNEL positive results wereobserved with respect to GrB-scFvMEL treated antigen-positive humanmelanoma A375-M cells at 48-hr but not with respect to GrB-scFvMELtreated antigen-negative human breast cancer SKBR-3-HP cells. Thisindicated that GrB-scFvMEL could specifically target antigen-positivemelanoma cells and induce cell apoptosis.

The effect of GrB-vegf121 on PAE/Flk-1 vs. PAE/Flt-1 cells by TUNELAssay was determined. VEGFR2/KDR was over-expressed on PAE/Flk-1 but notPAE/Flt-1 cell surface. VEGF121 specifically targeted VEGFR2/KDR,delivering GrB into PAE/Flk-1 cells. TUNEL Assay positive results wereobserved with respect to GrB-vegf121 treated PAE/Flk-1 at 24 hr and 48hr but not with respect to GrB-vegf121 treated PAE/Flt-1. GrB-vegf121induced PAE/Flk-1 cell apoptosis.

Example 33 Internalization of GrB/Vegf121 into PAE Cells byImmunofluorescence Microscopy

This example is directed to an internalization analysis of GrB/VEGF121by immunofluorescence microscopy. Cells were treated as follows. Cellswere plated in 16-well chamber slides (Nunc), 1×10⁴ cells per well,incubated for overnight at 37° C./5% CO₂ conditions. Cells were treatedwith 200 nM of GrB/VEGF121 for 4 h and then washed briefly with PBS. Thecell surface was stripped by incubations for 10 min with glycine buffer(500 mM NaCl, 0.1 M glycine, pH 2.5), neutralized for 2 min with 0.5 MTris, pH 7.4, and washed briefly with PBS.

Immunofluorescent staining occurred as follows. Cells were fixed in 3.7%formaldehyde for 15 min at RT, followed by a brief rinse with PBS andthen permeabilization for 10 min in PBS containing 0.2% Triton X-100.They were then washed three times with PBS. Samples were incubated with3% BSA for 1 h at RT to block nonspecific binding sites, then incubatedwith mouse anti-granzyme B antibody (1:100 dilution) at RT for 1 h,followed by washing three times with PBS. The samples were incubatedwith fluorescein isothiocyanate (FITC)-coupled anti-mouse IgG (Sigma)(1:100 dilution) at RT for 1 h and then washed three times with PBS. Thewalls and gaskets were removed carefully. After air drying, the slidewas mounted and analyzed under a fluorescence microscope.

The results showed that the GrB moiety of GrB/VEGF₁₂₁ was delivered intothe cytosol of PAE/flk-1 but not into that of PAE/flt-1 cells after 4 htreatment.

Example 34 Grb/Vegf121 Induces Apoptosis on PAE/Flk-1 Cells Detected byTunel Assay

Cleavage of genomic DNA during apoptosis may yield double-stranded, lowmolecular weight DNA fragments as well as single strand breaks (nicks)in high molecular weight DNA. Those DNA strand breaks can be identifiedby labeling free 3′-OH termini with modified nucleotides in an enzymaticreaction. This method uses terminal deoxynucleotidyl transferase (TdT),which catalyzes polymerization of nucleotides to free 3′-OH DNA ends ina template-independent manner, to label DNA strand breaks. Incorporatedfluorescein is detected by anti-fluorescein antibody Fab fragments fromsheep conjugated with alkaline phosphatase (AP). After substratereaction, stained cells can be analyzed under light microscope.

Cell treatments were as follows. Cells were plated onto 16-well chamberslides, 2500 cells/well, incubated for overnight at 37° C./5% CO₂conditions. Cells were treated with fusion protein GrB-vegf121 at I.C.₅₀concentration for different times (24, 48 hr, etc) and washed brieflywith PBS.

The TUNEL Assay was as follows. Cells were fixed with 3.7% formaldehydeat room temperature for 20 min, followed by rinsing with PBS andpermeabilization with 0.1% Triton X-100, 0.1% sodium citrate on ice for2 min. They were then washed with PBS twice. Cells were incubated withTUNEL reaction mixture at 37° C. for 60 min, followed by incubation withConverter-AP at 37° C. for 30 min, and finally reacted with Fast Redsubstrate solution at room temperature for 10 min. After final washstep, the slides were mounted in mounting medium and analyzed underlight microscope.

The results indicated that VEGFR2/KDR over-expressed on PAE/Flk-1 butnot the PAE/Flt-1 cell surface. VEGF121 specifically targetedVEGFR2/KDR, delivering GrB into PAE/Flk-1 cells. TUNEL Assay positiveresults were demonstrated for GrB/VEGF₁₂₁-treated PAE/Flk-1 at 24 hr and48 hr but not on GrB/VEGF121-treated PAE/Flt-1. Thus, GrB/VEGF 121induced PAE/Flk-1 apoptosis.

Example 35 Cytochrome C Release of PAE Cells Treated with GrB/Vegf121

Cytochrome c plays an important role in apoptosis. The protein islocated in the space between the inner and outer mitochonial membranes.An apoptotic stimulus triggers the release of cytochrome c from themitochondria into the cytosol where it binds to Apaf-1. The cytochromec/Apaf-1 complex activates caspase-9, which then activates caspase-3 andother downstream caspases.

Materials and methods for the cytochrome c release apoptosis assay:(from Oncogene Research Products; San Diego, Calif.) was as follows.PAE/flk-1 cells and PAE/flt-1 cells (5×10⁷) were treated withGrB/VEGF121 at concentrations of 0.1 nM and 20 nM for 24 h. Cells werecollected. After washing cells with 10 ml of ice-cold PBS, the cellswere resuspended with 0.5 ml of 1× cytosol extraction buffer mixcontaining DTT and Protease Inhibitors, and incubated on ice for 10 min.Cells were homogenized in an ice-cold glass homogenizer. The homogenatewas transferred to a 1.5 ml microcentrifuge tube and centrifuged at 700μg for 10 min at 4° C. The supernatant was transferred to a fresh 1.5 mltube and centrifuged at 10,000×g for 30 min at 4° C. Supernatant wascollected as a cytosolic fraction. The pellets were resuspended in 0.1ml mitochondrial extraction buffer mix containing DTT and proteaseinhibitors, vortexed for 10 seconds, and saved as a mitochondrialfraction. Protein concentrations were determined by using Bio-RadLaboratories, Inc. (Hercules, Calif.) Bradford Protein Assay. 10 μg ofeach cytosolic and mitochondrial fraction isolated from non-treated andtreated cells were loaded on a 15% SDS-PAGE, followed by standardWestern blot procedure and probing with cytochrome c antibody (1 μg/ml).

For the cytochrome c release apoptosis assay, PAE cells were treatedwith GrB/VEGF121 at different concentrations for 24 h. A highly enrichedmitochodrial fraction was isolated from the cytosol. Cytochrome ctranslocation from mitochondria into cytosol during apoptosis wasdetermined by western blotting using cytochrome c antibody. FIG. 22shows cytochrome c release on PAE/flk-1 but not on PAE/flt-1 cells afterGrB/VEGF121 treatment.

Example 36 Bax Translocation of PAE Cells after GrB/Vegf121 Treatment

Bax, a 21 kDa protein with extensive amino acid homology with Bcl-2, isvariably expressed by different cells. Bax as a pro-apoptotic member ofthe Bcl-2 family showed some structural similarities with pore-formingproteins. Hence, it is believed that Bax can form transmembrane poresacross the outer mitochondrial membrane, which leads to a loss ofmembrane potential. The localization of Bax has been shown to changefrom the cytosol to the mitochondria upon the receipt of an apoptoticstimulus.

Isolation of the cytosolic fraction and mitochondrial fraction wasperformed using an Oncogene Research Products kit (Cat # QIA87; SanDiego, Calif.). Protein concentrations were determined by using Bio-RadLaboratories, Inc. (Hercules, Calif.) Bradford Protein Assay. 10 μg ofeach cytosolic and mitochondrial fraction isolated from non-treated andtreated cells were loaded on a 12% SDS-PAGE, and then a standard Westernblot procedure was performed and probed with Bax antibody (Santa CruzBiotechnology, Inc.; Santa Cruz, Calif.; 1:200 dilution).

Western blotting analysis was performed of Bax expression on PAE cellsafter GrB/VEGF121 treatment for 24 h. The results showed that thelocalization of Bax changed from the cytosol to the mitochondria onPAE/flk-1 but not on PAE/flt-1 cells after treatment with GrB/VEGF121 atthe concentration of 20 nM (I.C.₅₀). FIG. 23 shows that Bax increased inmitochondria and decreased in cytosol on PAE/flk-1 cells afterGrBNEGF₁₂₁ treatment for 24 h at 20 nM concentration, indicating thatBax translocated from the cytosol to the mitochondria during apoptosis.

Example 37 Internalization of GrB/scFvMEL into A375-M Cells byImmunofluorescence Microscopy

Internalization analysis of GrB/scFvMEL by immunofluorescence microscopyutilized the following methods. Cells were plated in 16-well chamberslides (Nunc), 1×10⁴ cells per welt, and incubated for overnight at 37°C./5% CO₂ conditions. Cells were treated with 100 nM of GrB/scFvMEL for1 h and 6 h, then washed briefly with PBS. Cell surface was stripped byincubations of 10 min with glycine buffer (500 mM NaCl, 0.1 M glycine,pH 2.5), neutralized for 2 min with 0.5 M Tris, pH 7.4, washed brieflywith PBS.

For immunofluorescent staining, cells were fixed in 3.7% formaldehydefor 15 min at RT, followed by a brief rinse with PBS and thenpermeabilization for 10 min in PBS containing 0.2% Triton X-100; thecells were then washed three times with PBS. Samples were incubated with3% BSA for 1 h at RT to block nonspecific binding sites, then incubatedwith mouse anti-granzyme B antibody (1:100 dilution) at RT for 1 hfollowed by washing three times with PBS. The samples were incubatedwith fluorescein isothiocyanate (FITC)-coupled anti-mouse IgG (Sigma)(1:100 dilution) at RT for 1 h, washed three times with PBS. The wallsand gaskets were removed carefully. After air drying, the slide wasmounted and analyzed under light and fluorescence microscope.

The results showed that GrB moiety of GrB/scFvMEL was delivered into thegp240 antigen-positive A375-M cells by scFvMEL binding to gp240 antigen.

Example 38 GrB/scFvMEL Induces Apoptosis on A375-M Cells Detected byTunel Assay

Cells (3000 cells per well) were treated with GrB/scFvMEL at I.C.₅₀concentration for different times (16 h, 24 h) and washed briefly withPBS. Cells were fixed with 3.7% formaldehyde at room temperature for 20min, followed by rinsing with PBS and permeabilization with 0.1% TritonX-100, 0.1% sodium citrate on ice for 2 min. They were then washed withPBS twice. Cells were incubated with TUNEL reaction mixture at 37° C.for 60 min. After a final wash step, the cells were analyzed underfluorescence microscopy.

The results demonstrated that GrB/scFvMEL induced apoptosis onantigen-positive A375-M cells but not on antigen-negative SKBR3-HP cellsafter treatment for 16 h and 24 h.

Example 39 Cytochrome C Release in A375-M Vs. SKBR3-Hp Cells Treatedwith GrB/scFvMEL

Cytochrome c release apoptosis assay was performed as described(Oncogene Research Products; San Diego, Calif.; Cat# QIA87). Thematerials and methods were as follows. Human melanoma A375-M cells andhuman breast cancer SKBR3-HP cells (5×10⁷) were treated with GrB/scFvMELat concentrations of 5 nM and 50 nM for 24 h. Cells were collected.After washing cells with 10 ml of ice-cold PBS, cells were resuspendedwith 0.5 ml of 1× cytosol extraction buffer mix containing DTT andprotease inhibitors, incubate on ice for 10 min. Cells were homogenizedin an ice-cold glass homogenizer. Homogenate was transferred to a 1.5 mlmicrocentrifuge tube, and centrifuge at 700×g for 10 min at 4° C. Thesupernatant was transferred to a fresh 1.5 ml tube, and centrifuged at10,000×g for 30 min at 4° C. Supernatant was collected as a cytosolicfraction. The pellet was resuspended in 0.1 ml Mitochondrial ExtractionBuffer Mix containing DTT and protease inhibitors, vortexed for 10seconds, and saved as a mitochondrial fraction. Protein concentrationswere determined by using Bio-Rad Laboratories, Inc. (Hercules, Calif.)Bradford Protein Assay. 10 μg of each cytosolic and mitochondrialfraction isolated from non-treated and treated cells were loaded on a15% SDS-PAGE, and then a standard Western blot procedure was performedand probed with cytochrome c antibody (1 μg/ml).

Cytochrome c release apoptosis assay: A375-M and SKBR3-HP cells weretreated with GrB/scFvMEL at different concentrations for 24 h. Themitochondrial fraction was isolated from the cytosol, and thencytochrome c release from mitochondria into cytosol during apoptosis wasdetermined by western blotting using cytochrome c antibody. FIG. 24indicates that cytochrome c release on A375-M but not on SKBR3-HP cellsafter GrB/scFvMEL treatment.

Example 40 GrB/Vegf₁₂₁ Induces DNA Laddering on PAE/Flk-1 Cells

A DNA laddering assay procedure was followed to analyze influence ofGrB/VEGF121 on PAE/FLK-1 cells.

PAE cells (2×10⁶) were treated with GrB/VEGF121 at the I.C.₅₀concentration for 24 h. Cells were briefly washed with PBS and thenharvested by trypsinization, followed by centrifugation at 200×g for 5min. Cells were resuspended in 1 ml of PBS, transferred into 10 ml ofice-cold 70% ethanol and stored at −20° C. for 24 h or longer. Fixedcells were centrifuged at 800×g for 5 min, and the ethanol was removedthoroughly. The cell pellets were resuspended in 40 μl phosphate-citratebuffer (PCB), consisting of 192 parts of 0.2 M Na₂HPO₄ and 8 parts of0.1 M citric acid (pH 7.8) and incubated at RT for at least 30 min.After spinning cells down at 1000×g for 5 min, the supernatant wastransferred to a new tube. To samples were added 3 μl of 0.25% NonideNP-40 and 3 μl of RNase (1 mg/ml) and incubated for 30 min at 37° C.,followed by addition of 3 μl of proteinase K (1 mg/ml) and incubationfor another 30 min at 37° C. An 1.5% agarose gel was run to detect DNAby ethidium bromide under UV light.

FIG. 25 shows that GrB/VEGF121 induces DNA laddering on PAE/flk-1 butnot on PAE/flt-1 cells.

Example 41 ELISA of GrB/scFvMEL on gp240 Ag-Positive A375-M Vs gp240Ag-Negative T-24 Cells Detected by GrB Mouse MAB

Binding activity of GrB/scFvMEL to gp240 antigen-positive human melanomaA375-M vs gp240 antigen-negative human bladder cancer T-24 cells wasanalyzed by ELISA

Ninety six-well plates coated with 50,000 cells per well of A375-M orT-24 cells were blocked by 5% BSA, and the cells were then reacted withGrB/scFvMEL at various concentration for 1 h at RT. After washing, thesamples were incubated with GrB mouse monoclonal antibody (1 μg/ml) atRT for 1 h, followed by HRP-goat anti-mouse IgG. Then substrate2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) solutionwith 1 μl/ml of 30% H₂O₂ added. Absorbance at 405 nm was measured after30 min.

FIG. 26 shows that GrB/scFvMEL could specifically bind to Ag-positiveA375-M but not bind to Ag-negative T-24 cells, indicating there wasbinding activity of scFvMEL moiety of the fusion GrB/scFvMEL.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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All of the methods and compositions disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentsthat are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. The method of claim 5,wherein the amino acid sequence of said granzyme B is SEQ ID NO:60. 5.The method of claim 6, wherein the amino acid sequence of said granzymeB is at least 100 contiguous amino acids from SEQ ID NO:60.
 6. Themethod of claim 7, wherein the amino acid sequence of said granzyme B isat least 75 contiguous amino acids from SEQ ID NO:60.
 7. The method ofclaim 38, wherein the amino acid sequence of said granzyme B is at least40 contiguous amino acids from SEQ ID NO:60.
 8. The method of claim 7,wherein SEQ ID NO:60 further comprises an N-terminal extensioncomprising SEQ ID NO:61.
 9. The method of claim 7, wherein the firsttwenty amino acids are absent from SEQ ID NO:60.
 10. The method of claim37, wherein said granzyme is granzyme A.
 11. The method of claim 12,wherein the amino acid sequence of said granzyme A is SEQ ID NO:25. 12.The method of claim 13, wherein the amino acid sequence of said granzymeA is at least 100 contiguous amino acids from SEQ ID NO:25.
 13. Themethod of claim 14, wherein the amino acid sequence of said granzyme Ais at least 75 contiguous amino acids from SEQ ID NO:25.
 14. The methodof claim 10, wherein said granzyme A is at least 40 contiguous aminoacids from SEQ ID NO:25.
 15. The method of claim 36, wherein saidcell-specific targeting moiety is a cytokine, an antibody, a ligand, ora hormone.
 16. The of claim 15, wherein said ligand is VEGF.
 17. Themethod of claim 16, wherein said VEGF is vegf121.
 18. The method ofclaim 63, wherein said antibody is a single chain antibody.
 19. Themethod of claim 18, wherein said single chain antibody is scFvMEL. 20.The method of claim 36, wherein said granzyme is granzyme B and saidcell-specific targeting moiety is vegf121.
 21. The method of claim 36,wherein said granzyme is granzyme B and said cell-specific targetingmoiety is scFvMEL.
 22. The method of claim 36, further comprising alinker.
 23. The method of claim 22, wherein the linker comprises SEQ IDNO:50, SEQ ID NO:51, or SEQ ID NO:52.
 24. The method of claim 36,wherein the polypeptide is encoded by a recombinant polynucleotide.25.-35. (canceled)
 36. A method of inducing apoptosis in a cell,comprising administering to said cell an effective amount of a chimericpolypeptide comprising a cell-specific targeting moiety and a granzyme.37. The method of claim 36, wherein said granzyme is granzyme A orgranzyme B.
 38. The method of claim 37, wherein said granzyme isgranzyme B.
 39. The method of claim 36, wherein said cell is in vivo.40. The method of claim 39, wherein said cell is in a human.
 41. Themethod of claim 36, wherein said cell-specific targeting moiety isscFvMEL and said granzyme is granzyme B.
 42. The method of claim 36,wherein said cell-specific targeting moiety is vegf121 and said granzymeis granzyme B. 43.-49. (canceled)
 50. The method of claim 49, furtherdefined as a treating a disease in an individual, comprising the stepsof administering to said individual a therapeutically effective amountof a composition, wherein the composition comprises the chimericpolypeptide together with a pharmaceutical carrier.
 51. The method ofclaim 50, wherein said pharmaceutical carrier comprises a lipid.
 52. Themethod of claim 50, wherein said disease is cancer, diabetes, arthritis,or inflammatory bowel disease, atherosclerosis, or diabetic retinopathy.53. The method of claim 52, wherein said disease is cancer. 54.-59.(canceled)
 60. The method of claim 50, wherein said administration isintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, by inhalation, byinjection, by infusion, by continuous infusion, by localized perfusionbathing target cells directly, via a catheter, via a lavage, in a creme,or in a lipid composition.
 61. The method of claim 50, furthercomprising administering to said individual an anti-inflammatorycomposition, chemotherapy, surgery, radiation, hormone therapy, or genetherapy.
 62. The method of claim 53, wherein the cancer is wherein thecancer is melanoma, non-small cell lung, small-cell lung, lung,hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue,leukemia, neuroblastoma, head, neck, breast, pancreatic, prostate,renal, bone, testicular, ovarian, mesothelioma, cervical,gastrointestinal, lymphoma, brain, colon, sarcoma or bladder cancer. 63.The method of claim 50, wherein the cell-specific targeting moiety is anantibody.
 64. The method of claim 63, wherein the antibody is a singlechain antibody, a CDR, a monoclonal antibody, heteroconjugate antibody,chimeric antibody, or a polyclonal antibody.
 65. The method of claim 63,wherein the antibody is a human antibody or a humanized antibody. 66.The method of claim 63, wherein the antibody specifically binds Neu orgp240.
 67. The method of claim 50, wherein the cell-specific targetingmoiety is epidermal growth factor, fibroblast growth factor,insulin-like growth factor, or platelet-derived growth factor.
 68. Themethod of claim 36, wherein the granzyme is positioned N-terminal withrespect to said cell-specific targeting ligand.
 69. The method of claim36, wherein the granzyme is positioned at the N-terminus of the chimericpolypeptide.
 70. The method of claim 36, wherein the granzyme ischemically conjugated to the cell-specific targeting moiety.